membrane androgen receptor activation triggers pro-apoptotic … · 2019-10-30 · membrane...
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Membrane androgen receptor activation
triggers pro-apoptotic responses in vitro
and in vivo and blocks migration in colon
cancer
Dissertation
der Mathematisch-Naturwissenschaftlichen Fakultät
der Eberhard Karls Universität Tübingen
zur Erlangung des Grades eines
Doktors der Naturwissenschaften
(Dr. rer. nat.)
vorgelegt von
Shuchen Gu
aus Guizhou, V.R.China
Tübingen
2011
Tag der mündlichen Qualifikation: 31.08.2011
Dekan: Prof. Dr. Wolfgang Rosenstiel
1. Berichterstatter: Prof. Dr. F. Lang
2. Berichterstatter: Prof. Dr. Friedrich Goetz
Acknowledgments
It is with immense pleasure, I record my humble gratitude to my research guide
Prof. Dr. Florian Lang, for his expert guidance and critical review throughout the
work. My sincere and heart felt thanks for his abundant encouragement.
I am very grateful to my Ph.D supervisor Prof. Dr. Christos Stournaras for his
invaluable guidance, endless help, support, thought provoking suggestions and
stimulating discussions during the progress of the work. I would also like to
thank for his valuable input and suggestions for improving this dissertation.
I would also like to thank Prof. Fritz Götz for evaluating my work and for this
great help at my enrollment and in finalizing my thesis. And I would like to thank
him for giving me an opportunity to present the dissertation at the Faculty of
Biology, Eberhard Karls Universität Tübingen, Germany.
My heartfelt thanks to my colleague Dr. Michael Föller for their help and
assistance in organizing the lab work. His friendliness, patience made our lab
an exciting place for me to work. My heart felt thanks to my colleague Eva-
Maria Gehring, Omaima Nasir, Rexhep Rexhepaj, Hasan Mahmud, Nicole
Matzner, Diwakar Bobbala, Syed Qadri for their help, suggestions support and
friendship.
I would like to thank all other colleagues of the Institute of Physiology for making
the institute, a good place to work.
I dedicate my thesis to my loving parents for being a constant source of
inspiration for my work. I am greatly indebted for their support, constructive
criticism, prayers and endless love in my life.
Abstract
The classical intracellular androgen receptors (iAR) mediate genomic androgen
signals, which take at least more than half an hour. However, the rapid or non-
genomic action of androgens takes only seconds to few minutes and involves
the activation of androgen membrane binding sites. Although the molecular
identity of those membrane binding sites remains still unknown, their expression
has been reported in many cell types, including various tumor cells. Activation of
membrane androgen receptors (mAR) in prostate and breast cancer cells has
been implicated in the regulation of cell growth, motility and apoptosis. Here we
analyzed mAR expression and function in colon cancer. Using fluorescent mAR
ligands we showed specific membrane staining in mouse colon tumor tissues
and in iAR silenced Caco2 cell lines. Stimulation of colon-mAR by testosterone-
albumin-conjugates induced rapid actin and tubulin cytoskeleton reorganization
and generated apoptotic responses, even in the presence of anti-androgens.
We showed that long-term activation of mAR in Caco2 cell lines down-regulated
the activity of PI-3K and Akt and induced de-phosphorylation/activation of the
pro-apoptotic Bad. Treatment of APCmin/+ mice significantly decreased the
expression of p-AKT and p-Bad levels in tumor tissue. Moreover, mAR
activation resulted in a 65% reduction of tumor incidence in chemically induced
Balb/c mice colon tumors and an 80% reduction of tumor incidence in APCmin/+
mice colon tumors. Furthermore, mAR activation strongly inhibited Caco2 cell
migration. In accordance with this, vinculin, a protein controlling cell adhesion
and actin reorganization, was effectively phosphorylated upon mAR activation.
Phosphorylation inhibitors genistein and PP2 inhibited actin reorganization and
restored motility. Moreover, blocking actin reorganization by cytochalasin B and
silencing vinculin by appropriate siRNA’s restored the migration potential. From
these results we conclude that mAR activation inhibits the pro-survival signals
Akt/Bad in vitro and in vivo, induces potent proapoptoric responses and blocks
migration of colon cancer cells via regulation of vinculin signaling and actin
reorganization. Our results point to a central role of mAR in the induction of anti-
tumor responses in colon cancer.
Zusammenfassung
Die klassischen intrazellulären Androgenrezeptoren (iAR) vermitteln die
genomische Androgenwirkung, die wenigstens 30 Minuten Zeit erfordert. Im
Gegensatz hierzu benötigen die schnellen, nichtgenomischen Androgeneffekte
nur einige Sekunden bis wenige Minuten durch die Aktivierung von
Androgenbindungsstellen in der Zellmembran. Derartige membranständige
Androgenbindungsstellen wurden schon in vielen Zelltypen inklusive
Tumorzellen nachgewiesen, obwohl die molekulare Identität dieser
Bindungsstellen noch immer unbekannt ist. Die Aktivierung dieser
membranständigen Androgenrezeptoren (mAR) steht im Zusammenhang mit
der Regulation von Zellwachstum, Motilität und Apoptose. Mit der vorliegenden
Arbeit wurde die Expression und Funktion von mAR bei Kolonkarzinomen
untersucht. Mithilfe fluoreszierender mAR-Liganden konnten membranständige
Rezeptoren spezifisch angefärbt werden in Kolonkarzinomgewebe der Maus
und in Caco2-Zellen, die nicht über iAR verfügen. Die Stimulierung von mAR in
Dickdarmgewebe durch Testosteron-Albumin-Konjugate führte zu einer raschen
Reorganisation des Aktin- und Tubulinnetzwerkes und löste Apoptose selbst in
Anwesenheit von Antiandrogenen aus. Die längeranhaltende Aktivierung von
mAR in Caco2-Zellen führte zu verminderter PI-3- und Akt-Kinaseaktivität und
zur Dephosphorylierung und mithin Aktivierung von proapoptotischem Bad. Eine
entsprechende Behandlung von APCmin/+-Mäusen verringerte die Expression
von p-Akt und von p-Bad signifikant in Tumorgewebe. Darüber hinaus führte die
Aktivierung von mAR zu einer Verminderung der Tumorinzidenz um 65% bei
chemisch induzierten Dickdarmtumoren von Balb/c-Mäusen und um 80% bei
APCmin/+-Mäusen. Weiterhin hemmte die mAR-Aktivierung die Zellmigration
von Caco2-Zellen stark. In Übereinstimmung mit diesem Befund war Vinculin,
ein Protein, das Zelladhäsion und Aktinreorganisation reguliert, nach mAR-
Aktivierung deutlich phosphoryliert. Die Phosphorylierungsinhibitoren Genistein
und PP2 hemmten die Aktinreorganisation und reaktivierten die Zellmotilität.
Zusätzlich konnte die Blockade der Aktinreorganisation durch Cytochalasin B
oder die auf siRNA basierende Herunterregulation von Vinculin das
Migrationspotential der Zellen wiederherstellen. Aus diesen Daten kann
geschlossen werden, dass die Aktivierung von mAR die überlebensfördernden
Akt/Bad-Signale in vitro und in vivo hemmt, wirkungsvolle proapoptotische
Zellantworten induziert und die Migration von Kolontumorzellen über die
Regulierung des Vinculin-Signalweges und der Aktinreorganisation blockiert.
Diese Erkenntnisse deuten auf eine zentrale Rolle von mAR für die Induktion
von Antitumorantworten bei Kolonkarzinom.
Content
Acknowledgments .............................................................................................. 3
Content ............................................................................................................... 7
1. Introduction ...........................................................................................11
1.1 Androgens .........................................................................................11
1.1.1. Intracellular Androgen Receptors ........................................... 12
1.1.2. Membrane Androgen Receptor .............................................. 17
1.2 Colon cancer .................................................................................... 21
1.2.1. Epidemiology of colorectal cancer .......................................... 21
1.2.2. Etiology of colorectal cancer .................................................. 21
1.2.3. Genes .................................................................................... 22
1.2.4. Colon cancer and steroid receptors ....................................... 23
1.3 Apoptosis ......................................................................................... 24
1.3.1. Morphological features of apoptosis ....................................... 24
1.3.2. Molecular mechanisms of apoptosis signaling pathways ....... 25
1.3.3. Apoptosis responses by AKT pathway ................................... 26
1.3.4. Apoptosis responses by Bad .................................................. 27
1.3.5. Apoptosis responses by actin polymerasion .......................... 29
1.4 Cell Migration ................................................................................... 30
1.4.1. PI3K/Akt signaling pathways in cell migration ........................ 31
1.4.2. Vinculin and cytoskeleton protein Actin in cell migration ........ 32
1.5 Cell and animal model of colorectal cancer ...................................... 35
2. Aims of the studies............................................................................... 37
3. Materials and methods ......................................................................... 38
3.1 Materials .......................................................................................... 38
3.1.1. Chemical and biological reagents .......................................... 38
3.1.2. Equipment .............................................................................. 40
3.2 Methods ........................................................................................... 41
3.2.1. Cell culture ............................................................................. 41
3.2.2. Preparation of steroid solution ................................................ 41
3.2.3. In vivo animal experiment....................................................... 41
3.2.4. Immunofluorescence analysis and confocal laser scanning
microscopy ........................................................................................... 43
3.2.5. Immunoprecipitation and Westen blotting .............................. 44
3.2.6. Measurement of G/total actin ratio by Triton X-100 fractionation
45
3.2.7. Matrigel and transwell assay .................................................. 46
3.2.8. Wound healing assay ............................................................. 47
3.2.9. siRNA experiments ................................................................. 47
3.2.10. TUNEL assay ......................................................................... 48
3.2.11. APOPercentage apoptosis assay ........................................... 48
3.2.12. Statistical analysis .................................................................. 49
4.
RESULTS
............................................................................................ 50
4.1 mAR expression in colon cancer cell lines ....................................... 50
4.2 mAR expression in 2 different colon cancer animal models ............. 52
4.3 mAR activation by testosterone-HSA was followed by extensive
reduction of tumor incidence in vivo ........................................................... 53
4.4 p-Akt and p-Bad are downregulated in colon tumor tissues treated by
testosterone-HSA ....................................................................................... 55
4.5 mAR stimulation inhibits Akt activity and induces Bad de-
phosphorylation in Caco2 but not in IEC06 cells ........................................ 57
4.6 mAR activation triggered rapid actin and tubulin reorganization in
colon cancer cells ....................................................................................... 59
4.6 mAR activation inhibits cell motility in colon cancer cells ................. 63
4.7 mAR activation triggers vinculin phosphorylation ............................. 66
4.8 Vinculin is necessary for actin reorganization and migration of mAR
stimulated Caco2 cells ............................................................................... 69
5. Discussion ........................................................................................... 74
5.1 Membrane androgen receptor activation in colon cancer triggers
pro-apoptotic responses in vitro and in vivo ......................................... 75
5.2 Membrane androgen receptor activation blocks migration ........ 78
6. Conclusions ......................................................................................... 82
Reference ......................................................................................................... 83
Abbreviations
3a-Diol 5a androstane-3a, 17b diol
5aR 5alpha reductase enzyme
AF-1 active activation function
AF-2 ligand-dependent activation function
AKR1C aldo-keto reductase
Apaf-1 apoptoic protease activating factor-1
APC adenomatous polyposis coli
AR androgen receptor
BSA bovine serum albumin
CAD caspaseactivated DNase
CAG encode polyglutamine
CAM calmodulin
cAMP cyclic adenosine monophosphate
CIN chromosomal instability
CYP cytochromes P450
DBD DNA-binding domain
DD death domains
DED death effector domains
DHT dihydrotestosterone
EGF epidermal growth factor
ER estrogen receptor
ERK extracellular-signal regulated kinase
FADD Fas-Associated protein with Death Domain
FAK focal adhesion kinase
FAP familial adenomatous polyposis
FGF fibroblast growth factor
GABA gamma-aminobutyric aci
GDP guanosine diphosphate
GGC polyglycine
GP G-protein
GPCR G-protein coupled receptor
GTP guanosine triphosphate
HNPCC hereditary nonpolyposis colon cancer
HSD hydroxysteroid dehydrogenase
IAPs inhibitor of apoptosis proteins
iAR intracellular androgen receptor
IBD inflammatory bowel disease
ICAD inhibitor of caspase-activated DNase
ICE interleukin-1β-converting enzyme
IGF1 Insulin-like growth factor 1
IP3 inositol 1,4,5-triphosphate
LBD C-terminal ligand-binding domain
MAPK mitogen-activated protein kinase
mAR membrane associated androgen receptor
MEK MAPK/ERK kinase
MMR mismatch repair
MSI microsatellite instability
PARP poly ADP-ribose polymerase
PI3K phosphatidylinositol 3-kinase
PKA protein kinase A
PKC protein kinase C
PLC phospholipase C
PTK protein tyrosine kinase
ROCKI Rho-associated coiledcoil forming kinase I
SEER Surveillance, Epidemiology and End Results
SH2 Src homology domain 2
SH3 Src homology domain 3
SHBG steroid hormone-binding globulin
SHBGR steroid hormone-binding globulin receptor
SHC SH2 Containing Protein
SMAD4 Mothers against decapentaplegic homolog 4
SR sarcoplasmic reticulum
T testosterone
TAD N-terminal transactivation domain
TGFß transforming growth factor-β
TIF2 Transcription Intermediary Factor-2
TP53 tumor protein P53
TRADD TNFRSF1A-associated via death domain
VEGF vascular endothelial growth factor
1. Introduction
1.1 Androgens
Androgens are important male sex steroid hormones. They have many
physiological roles leading to the male characteristics and other phenotypes.
The major circulating androgen in human tissue is testosterone, which is
synthesized mainly by Leydig cells in testis. The effects of testosterone can be
classified as virilizing and anabolic. Anabolic effects will make muscle more
mass and strength, increase bone density and strength, and stimulate the linear
growth and bone maturation. Virilizing effects maturate the sex organs,
particularly the penis and the formation of the scrotum in unborn children. And
after birth the virilizing effects include a deepening of the voice, growth of the
beard and axillary hair. Most of these fall into the category of male secondary
sex characteristics. There is another potent androgen, dihydrotestosterone
(DHT) in addition to testosterone. It is synthesized mostly in peripheral tissue.
The responsibility of DHT results in all of the male secondary sexual
characteristics such as deepening of the vocal chords, male hair patterns on the
body, hair on the face, oily, and male sexual drive and function. There are other
two weak androgens, which are dehydroepiandrosterone and androstenedione.
They are mostly synthesized in adrenal glands.
The synthesizing of Androgens is from steroidogenic pathways involving various
enzymes and many different intermediates. They are under the control of the
stringent regulation through the hypothalamus-pituitary-testis axial. A
biosynthetic pathway of androgen starts from cholesterol, which are functions as
the precursor. Two androgens, dehydroepiandrosterone and androstenedione
are mainly produced in adrenal tissue. Androstenedione is converted into
testosterone or estradiol mainly in testis and peripheral tissue. Testosterone is
further changed to the more potent 5ɑ-dihydrotestoterone by 5ɑ-reductase, or
converted into estradiol by aromatase. Enzymes involved in androgen synthesis
pathways are mainly proteins in the cytochromes P450 (CYP) and
hydroxysteroid dehydrogenase (HSD) families. [Chang C. 2002] In addition to
the controlled synthesis, androgens are removed from blood through a series of
well-orchestrated pathways. They are converted to other active metabolites first,
which then followed by sulfation and conjugation with glucuronic acid so as to
become more hydrophilic.
The function of androgen is very important. Any change in steps of androgen
synthesis will result in diseases. The deficient androgen synthesis often causes
male pseudohermaphroditism. Problems in the regulation of androgen synthesis
can also lead to male and female pseudohermaphroditism. [Guido M, Uta C.P.
2008] Many genetic diseases due to abnormal androgen secretion have been
described. These diseases usually arise due to mutations in the genes involved
in steroid metabolism. The cause of hormone-dependent tumors, e.g. prostate
cancer, is also due to the change of androgen secretion.
1.1.1. Intracellular Androgen Receptors
The intracellular androgen receptor (iAR) is also known as NR3C4 (nuclear
receptor subfamily 3, group C, member 4). It is a type of nuclear receptor which
is activated by binding of either the androgenic hormones testosterone or DHT.
As other members of the nuclear receptor superfamily, iAR has four major
functional regions: an N-terminal transactivation domain (TAD), a central DNA-
binding domain (DBD), a C-terminal ligand-binding domain (LBD), and a hinge
region connecting the DBD and LBD [Mangelsdorf DJ, et al. 1995].
The classic genomic model for steroid hormone action presumes that steroid
hormones can freely cross the Plasma Membrane, enter the cytoplasm, and
bind to activate specific iAR. The bound steroid receptors act as transcription
factors and bind as homodimers or heterodimers to specific DNA response
elements in target gene promoters, causing protein synthesis. [Guido M, Uta
C.P. 2008] iAR is a kinase substrate and downstream target of receptor-tyrosine
kinase (RTK), for example HER-2/neu, and G-protein coupled receptor (GPCR)
signalling which can both activate AR independently of androgen. [Nigel C.
Bennett, et al. 2010] This genomic-androgen effect typically takes at least
more than half an hour. There are also another two pathways activated will
active by iAR. One is binding with the SH3 domain of the tyrosine kinase c-Src
to active the MAPK pathway and influence the iAR-mediated transcription via
phosphorylation of receptor complexes. The other is binding to steroid
hormone-binding globulin (SHBG).The iAR and SHBG bound complexes can
activate SHBG receptor and lead to an increase in PKA activity. PKA may
influence the iAR-mediated transcription via alteration of phosphorylation status
of iAR and iAR coregulators. [Guido M, Uta C.P.2008] (Fig.1)
Figure 1: Androgen actions via intracellular androgen receptor.
(1) In the classical pathway. (2) Bound with the SH3 domain. (3) Bound to SHBG. Abbreviations:
T, testosterone; DHT, dihydrotestosterone; 5αR, 5alpha reductase enzyme; AR, androgen
receptor; PKA, protein kinase A; GP, G-protein; SH2, Src homology domain 2; SH3, Src
homology domain 3; PTK, protein tyrosine kinase; MAPK, mitogen-activated protein kinase;
SHBGR, steroid hormone-binding globulin receptor; cAMP, cyclic adenosine monophosphate.
The iAR activated target genes are an array of growth factor genes, e.g.
epidermal growth factor (EGF); fibroblast growth factor (FGF); Insulin-like
growth factor 1(IGF1); vascular endothelial growth factor (VEGF); transforming
growth factor-β (TGFβ). The ability of iAR to cross-talk with key growth factor
signaling events toward the regulation of cell cycle, apoptosis, and
differentiation outcomes in prostate cancer cells has been established. IGF,
FGF, VEGF, and TGFβ secreted by the prostate stromal cells activate their
receptors and interact with iAR signal axis. In prostate epithelial cells, the
androgenic signal engages secreted VEGF and TGFβ which affect the prostate
tumor microenvironment by inducing angiogenesis, stromal cell growth and
differentiation. EGF signaling encounters iAR signal in a tight control of multiple
pathways. Growth factor signaling may proceed via iAR signal and regulate the
downstream effectors of iAR regulating key cellular processes including
proliferation, differentiation, apoptosis, and survival of prostate cancer cells.
[Meng-Lei Zhu, Natasha Kyprianou. 2008]
Several studies show that some connections may exist between iAR and the
Wnt pathway. The interaction of iAR and the phosphatidylinositol 3-kinase/Akt
pathway has been demonstrated in prostate cancer cell lines. The tumor
suppressor PTEN, which inhibits the PI3K/Akt pathway, is frequently mutated in
prostate cancer. PTEN has been shown to modulate androgen-induced prostate
cancer cell growth and iAR-mediated transcription [Li P, et al. 2001; Wen Y et al.
2000]. A study using a synthetic PI3K inhibitor, and re-expression of PTEN in a
PTEN-null prostate cancer cell line shows that the involves the Wnt pathway:
GSK3 , a downstream effector of PI3K/Akt, also participates in the Wnt
pathway; PTEN phosphorylate and inactivate GSK3 , a downstream effector of
PI3K/Akt, via PI3K/Akt; GSK3 -dependent inactivation of cytoplasmic -catenin
is was subsequently attenuated; As a result, -catenin shuttled to the nucleus
and augmented ligand-stimulated transcription by iAR. [Sharma M, et al. 2001]
In addition to the transcriptional or genomic mode of activation by steroids,
androgens can also exert rapid, nongenomic effects. Similar to the non-genomic
action of other steroids, there are certain basic criteria for an androgen induced
response to be considered non-genomic in nature. The first criterion is speed.
The effect should occur in seconds to minutes. It is not long enough to allow
gene transcription or translation. Typically, gene transcription needs several
hours after steroid exposure, although the latency for transcription events has
been reported to be as short as 7.5 min. It has to take the additional time for
mRNA to be translated into proteins and for those proteins to be processed and
induce measurable responses. The cellular responses are changes in free
intracellular calcium, and activations of second messenger pathways. The
second criterion is membrane mediated. The response should include
embedding membrane or associating receptors or binding proteins. The action
can be induced even when the steroid is conjugated to molecules which prohibit
it from translocation to the nucleus when bound to a receptor. The most
common example is the use of testosterone (T) conjugated to large molecules
such as bovine serum albumin (BSA). The last is lacking transcription/
translation machinery activation. Experiments using cell lines either lacking the
necessary machinery for a genomic response or identifying androgen effects,
they are insensitive to inhibitors of transcription and translation. All this
demonstrated that certain steroid responses can be elicited in systems where
gene transcription or protein synthesis is was unlikely or impossible.
[C.D.Foradori, et al. 2008]
Figure 2: Non-genomic androgen actions via intracellular ion concentrations and
membrane fluidity.
(1) Androgen interacts with a mAR leading to the activation of L-type calcium channels through
an inhibitory G-protein (GP). (2) Androgen interacts with mAR leading to modulation of G-protein
activity and subsequent activation of phospholipase C (PLC). (3) DHT’s metabolite, 3a-Diol,
may interact with the GABAA receptor and lead to increases in intracellular calcium and thus
membrane potential. (4) Testosterone and its metabolites can interact with phospholipids in the
membrane bilayer to change membrane flexibility and subsequently alter the function of
sodium/potassium ATPase and calcium ATPase. Abbreviations: T, testosterone; 5aR, 5alpha
reductase enzyme; AKR1C, aldo-keto reductase; 3a-Diol, 5a androstane-3a, 17b diol; GABA,
gamma-aminobutyric acid; GP, G-protein; PKA, protein kinase A; PKC, protein kinase C; CAM,
calmodulin; PLC, phospholipase C; IP3, inositol 1,4,5-triphosphate; SR, sarcoplasmic reticulum;
MEK, MAPK/ERK kinase; ERK, extracellular-signal regulated kinase. [C.D.Foradori, et al. 2008]
Nongenomic steroid activity involves the rapid induction of conventional second
messenger signal transduction cascades. Nongenomic action of androgens can
occur through multiple receptors. Androgens activate cAMP and PKA through
membrane androgen receptor (mAR). Androgens also induce an elevation in
intracellular Ca2+ through mAR to a GPCR (G-Protein Coupled Receptor) by
activating an influx through nonvoltage-gated Ca2+ channels. The increasing of
intracellular calcium activates signal transduction cascades, which is included
PKA (Protein Kinase-A), PKC (Protein Kinase-C), and MAPKs (Mitogen-
Activated Protein Kinase). They can modulate the activity of the ARs and other
transcription factors. AR can also interact with the intracellular tyrosine kinase c-
Src, triggering c-Src activation. One of the targets of c-Src is the adapter protein
SHC (SH2 Containing Protein). It is an upstream regulator of the MAPK
pathway. The activation of AR are influenced by direct phosphorylation by
MAPK [Heinlein CA, Chang C. 2002]. In another side, AR phosphorylation by
ERK2 is associated with enhanced AR transcriptional activity and an increased
ability to recruit the coactivator ARA70.[Heinlein CA, Chang C. 2002] The SRC
family of transcriptional coactivators includes SRC1, SRC3, and TIF2
(Transcription Intermediary Factor-2). They are targets of MAPK
phosphorylation and result in an increased ability of these coactivators to recruit
additional coactivator complexes to the DNA-bound receptor. The nongenomic,
rapid stimulation of second messenger cascades by androgens may ultimately
exert biological effects through modulation of the transcriptional activity of AR or
other transcription factors. Those modulations may happen by direct
phosphorylation of transcriptional activators or their coregulators [Michels G,
Hoppe UC. 2008]. In the absence of AR’s cognate ligand the AR can also be
activated. Androgen can initiate by various growth factors.
1.1.2. Membrane Androgen Receptor
Scientific evidence accumulated in recent year’s points to the existence of
membrane androgen receptors (mARs), triggering rapid, non-genomic signals.
Although the exact molecular identity of mAR still remains unknown, non-
genomic androgen actions manifested within minutes have been reported in
various cell types including macrophages and T cells [Benten WP, et al. 1999;
Benten WP, et al. 1999], LNCaP [Kampa M, et al. 2002; Wang Z, et al. 2008],
T47D [Kampa M, et al. 2005], MCF7 [Kallergi G, et al. 2007], DU145 [Hatzoglou
A, et al. 2005; Papadopoulou N, et al. 2008a; Papadopoulou N, et al 2008b], C6
[Gatson JW, et al. 2006], PC12 [Alexaki VI, et al. 2006] or VSMC cells [Somjen
D, et al 2004]. These effects are clearly different from those manifested upon
activation of the intracellular androgen receptors (iARs) mediating genomic
androgen signals resulting in receptor dimerization, nuclear translocation and
subsequent activation of androgen-specific target genes.
In prostate cancer, expression of mAR in human tumor cells was initially
reported in iAR positive LNCaP cells [Kampa M, et al 2002] and iAR –deficient
DU145 cells [Hatzoglou A, et al 2005]. In LNCaP cells study, mAR activation
through testosterone-BSA conjugates induced rapid PSA release, fast actin
reorganization and additional cell responses like inhibition of cell growth and
induction of apoptosis [Hatzoglou A, et al 2005]. The molecular signaling
pathway starts from focal adhesion kinase (FAK). Initially, FAK was rapidly
phosphorylated and associated with the p85 subunit of the phosphoinositol-3-
Kinase (PI-3K). Following this association, the lipid kinase activity of PI-3K and
the tyrosine phosphorylation of its p85 regulatory subunit were significantly
induced by mAR stimulation. PI-3K activation was accompanied by the
downstream upregulation of the Rho small GTPases Cdc42, Rac1, RhoA and
RhoB. Rapid activation of these GTPases resulted in actin cytoskeleton
reorganization. Yet again, these effects were specific for mAR because three
different steroidal and non-steroidal iAR antagonists failed to block the
activation of this rapid signaling pathway [Papakonstanti, E. A., et al. 2003].
From these findings it was concluded that mAR activation induced potent
apoptotic regression in LNCaP prostate tumor cells controlled by
Rho/ROCK/actin signaling. Interestingly, while LNCaP prostate cancer cells
express functional iAR, the DU145 cell line expresses either nonfunctional iAR,
or is iAR-deficient. Therefore, DU145 cells fail to respond to iAR-regulated
androgen treatment.[ Alimirah, F., et al 2006] In this cell model, mAR stimulation
by testosterone or T-BSA conjugates induced potent actin reorganization,
inhibited cell motility and promoted apoptotic regression. [Papadopoulou N, et
al. 2008a] But the signaling pathway is different from LNCaP cells. Specifically,
mAR activation bypassed the FAK/ PI-3K signaling pathway, as FAK was shown
to be constitutively phosphorylated and mAR stimulation failed to further
activate the downstream effectors PI-3K and Rac. An alternative pathway
functionally distinct from the FAK/PI-3K/Rac signaling was described.
[Papadopoulou N, et al. 2008a] This pathway regulated actin reorganization, the
induction of apoptosis and the pro-apoptotic machinery. Indeed, long term down
regulation of the pro-survival PI-3K/Akt pathway became evident 12–24 h upon
mAR activation as indicated by the significant decrease of the phosphorylation
levels of PI-3K and Akt. Furthermore, inhibition of NF-jkB translocation and
increased FasL expression were documented, while increased caspase 3
activity was measured [Papadopoulou N, et al 2008b].
A B
Figure 3: mAR signaling in human prostate cancer cells
A) Non-genomic mAR signaling operating in iAR positive LNCaP human prostate cancer cells
regulating actin redistribution and apoptosis. Solid arrows indicate events that have been
experimentally proven. Dashed arrows indicate unidentified possible links. See text for details.
B) Early and late mAR signaling operating in iAR deficient DU145 human prostate cancer cells
regulating actin redistribution, downstream pro-apoptotic signaling, and migration. Solid arrows
indicate events that have been experimentally proven. Dashed arrows indicate unidentified
possible links. See text for details. [Papadopoulou N, et al 2009]
In breast cancer, it has been reported that mAR is expressed in T47D and
MCF7 human breast epithelial cancer cells. In T47D cells, specific and
saturable androgen receptors are present in the membrane and their activation
via TBSA conjugates resultes in cell death by apoptosis. [Kampa M, et al 2005]
Moreover, pharmacological inhibitors of MEK and p38 kinase were able to block
T-BSA induced apoptosis showing a functional implication of these pathways in
mAR-dependent apoptosis in T47D cells. However, in MCF7 cells, activation of
these receptors by T-BSA conjugates triggered a non-genomic signaling
pathway involving FAK and PI-3K phosphorylation and downstream activation of
the small GTPase Rac1, ultimately resulting in actin redistribution. Cell
migration experiments provided insights in the functional role of mAR
stimulation in MCF7 cells. But the activations of mAR did not induce any
apoptotic response in this kind of cells. [Kallergi G, et a.l 2007]
Figure 4: mAR signaling in breast epithelial cancer cells
Non-genomic mAR signaling operating in MCF7 breast epithelial cancer cells regulating actin
redistribution and cell motility. Solid arrows indicate events that have been experimentally
proven. Dashed arrows indicate unidentified possible links. [Papadopoulou N, et al 2009]
Taken together, these studies clearly established that functional mARs trigger
strong anti-tumorigenic effects in prostate and breast cancer cells, implying a
potential role of mAR as a novel target for the development of selective cancer
treatments [Papadopoulou et al., 2009]. It was shown that mAR activation
resulted in actin reorganization regulated by distinct mechanisms involving
small GTPases’ specific signaling cascades. [Papadopoulou N, et al 2008b]
Furthermore, it was shown that mAR activation induced potent apoptotic
regression of prostate cancer cells in vitro [Papadopoulou N, et al 2008a] and in
mouse xenografts in vivo and suppressed cell growth and motility. [Hatzoglou A,
et al 2005, Kampa et al 2006] In breast cancer, activation of mAR in MCF7
breast epithelial cancer cells regulates actin redistribution and cell motility.
[Papadopoulou N, et al 2009] However, it remained elusive whether mARs were
also expressed in other tumors and whether their activation could result in the
induction of anti-tumorigenic effects similar to the ones described in prostate
and breast cancer cells.
In my diploma thesis, by using either colon cancer tissues isolated from mice
xenograft tumors or two established colon cancer cell lines (Caco2 and HCT116
cells), the expression and function of functional role of mAR has been analyzed.
As a result, testosterone binding sites were expressed in the membrane of
colon cancer cells and qualify as bona fide membrane androgen receptors as
assessed by radioligand binding studies, Scatchard analysis and displacement
assays. The activation of those receptors with non permeable testosterone
derivatives induced pro-apoptotic responses. [Gu S, et al. 2009]. However, the
expression and regulation of pro-survival signals that may compensate mAR
induced cell death remained undefined. In addition, the profound anti-
tumorigenic mAR action has not been addressed in association with motility and
invasiveness. In my PhD work I addressed the anti-apoptotic signaling in mAR
expressing colon tumors in vitro and in vivo. Moreover, the migration potential of
colon tumor cells upon mAR stimulation was examined and the molecular
targets were analyzed in detail, implicated in cell motility regulation.
1.2 Colon cancer
1.2.1. Epidemiology of colorectal cancer
Colon cancer is considered a public health problem worldwide. It is also called
colorectal cancer (CRC) or large bowel cancer including cancerous growths in
the colon, rectum and appendix. With 655,000 deaths worldwide per year, it is
the third most common form of cancer and the second leading cause of cancer-
related death in the Western world. The incidence rates are higher in the
developed areas, such as Europe, North America and Australia. In Cancer
incidence of Europe 2006, it was estimated that 217400 and 195400 new colon
cancer cases occurred annually in men and women in Europe, ranking third in
the incidence in men and second in women. [Ferlay, J, et al.2007] It’s noticeable
that the incidence of colon cancer used to be low in some eastern countries,
such as Japan and China. However, in recent decades the incidence and
mortality of colon cancer in these countries increased sharply, which was
probably owing to their adoption of Western life style. The mortality of colon
cancer in Japan increased 5.5-folds in the second half of the 20th century
[Honda, T., et al. 1999]. The incidence of colon cancer in China doubled in the
past 30 years as well [Zhang, Y.Z., et al. 2005]. According to the Surveillance,
Epidemiology and End Results (SEER) Program database analysis, 5-year
survival rates have risen from 56.5% for patients diagnosed in the early 1980s
to as much as 63.2% for those diagnosed in the early 1990s and most recently
to 64.9%, a trend due mostly to earlier diagnosis and treatment [Ries LAG, et al.
2008]. One reason for the improving trend is that the prognosis for patients with
CRC is highly dependent on stage: 5-year survival rates are over 90% for
Dukes A, but only 5% for Dukes D. Unfortunately, only 10% of CRCs are
diagnosed early, most patients presenting themselves with the advanced
disease [Rockville, MD. 1998].
1.2.2. Etiology of colorectal cancer
The risk factors of CRC include age older than 50, inflammatory bowel disease
(IBD), high-fat low-vegetable diet, physical inactivity, smoking, alcohol and
genetic predeposition. [Benson AB 3rd. 2007; Jemal, A., et al 2007] About 20%
of CRC are familial, which means gene alterations may mediate the
development of CRC [Benson, 2007]. There are two key CRC related hereditary
diseases, familial adenomatous polyposis (FAP) and hereditary nonpolyposis
colon cancer (HNPCC). FAP is a rare autosomal dominant syndrome caused by
an inherited mutation in the APC gene [Strate LL, Syngal S. 2005]. It accounts
for approximately 1% to 2% of all CRC cases. HNPCC or termed as Lynch
syndrome [Lynch, P.M., Lynch, H.T. 1985], an inherited autosomal dominant
syndrome, is caused by inherited mutation in any one of five DNA mismatch
repair (MMR) genes and microsatellite instability [Benson AB 3rd. 2007].
HNPCC is predicted to account for 2% or less of all CRC cases [Aaltonen, L.A.,
et al. 1998].
1.2.3. Genes
There is much progress which has been made in understanding the molecular
mechanism of colorectal cancer. A progression from normal mucosa to
adenoma to carcinoma was supported by the demonstration of accumulating
mutations in genes as K-ras, adenomatous polyposis coli (APC), tumor protein
P53 (TP53), and deleted in DCC, all of which are thought to be of significance,
but are not able to successfully account for all colorectal cancers. There is
heterogeneity in the pathogenetic pathway leading to CRCs, and there are two
major tumorigenic pathways. The first is driven by chromosomal instability
(CIN), the progress of which involves both oncogenes and tumor-suppressor
genes including chromosomes 5q, 17p, and 18q [Fearon, E.R., Vogelstein, B.
1990; Gervaz, P., et al. 2001]. Chromosome 5q genes are responsible for APC,
17p for TP53, and 18q for DCC or Mothers against decapentaplegic homolog 4
(SMAD4). K-ras is the most common oncogene following this pattern. The
tumor-suppressor genes APC, TP53, and DCC/SMAD4 play important roles in
this sequential adenoma to carcinoma. Another genetic pathway may well be
depicted as a consequence of the alteration in mismatch repair (MMR) genes.
[Gervaz, P., et al. 2001] When the alteration happens in germinal cells, the
hereditary cancer known as hereditary nonpolyposis colorectal cancer (HNPCC)
occurs. When somatic cells are affected, microsatellite instability (MSI) would be
unavoidable. MSI is responsible for a subset of sporadic colorectal tumors.
[Feng-ying LI, Mao-de LAI 2008]
1.2.4. Colon cancer and steroid receptors
Although colon cancer is not a hormone-dependent tumor, the existence of sex
differences in colon cancer incidence was proposed several years ago. The
activity of steroid receptor plays a pivotal role in a well-controlled cascade of
signals, which maintains the mucosal architecture by the shedding of senescent
and apoptotic cells at the surface of the epithelium. The identification of a
functional interaction between the Wnt/APC pathway and steroid represents a
major goal for several laboratories [Mulholland, et al 2005]. β-Catenin activates
a growing number of steroid receptors, resulting in alterations of cell
proliferation and tumorigenesis. On the other hand, Wnt signaling appears to be
compromised by the action of some steroid receptors. It is also clear that steroid
receptors are regionally compartmentalized along the cryptvillus axis,
determining the switching on and off of transcription of particular genes with a
strong influence on cell fate. The mechanism for the influence of steroid
receptors on cell proliferation, differentiation and apoptosis in the gut is complex
and still under investigation. Also, the observed phenotypes after steroid
receptor activation or inhibition are sometimes contradictory. Steroid receptor
effects depend on the amount of agonists, the cell type and the mutational.
[Mulholland, et al. 2005] In estrogen receptor, ERβ, dependent or independent
of 17β-oestradiol activation has a crucial role in colonic cell homeostasis,
including modulation of proliferation and organized cell death. Absence of ERβ
results in increased cell turnover in the colonic mucosa. The specific ERβ
signaling confers protection against colonic mitogenesis. ERβ modulators might
provide options for prevention of colorectal cancer, but variance in signaling of
ERβ will complicate the development of treatments. Non-genomic activation of
mitogenic cell signaling cascades by oestrogen and clarification of receptor
independent coupling mediated by protein kinase C (PKC) δ provide other
opportunities for cancer treatment. [Kennelly R, et al. 2008] Colon cancer is also
related to androgen receptor. The selective binding between lithocholic acid and
iAR supports that diet-related endoluminal substance may play a role in cancer
development model. Moreover molecular alterations such as DNA damage or
mutation are the 1st event. [Berta, L., et al 2003] An association between the
iAR genotype and colorectal cancer has been observed. Although the
expression of iAR, implicated in tumorigenesis has been reported in colon
tumors [Slattery ML, et al 2005], the role of functional membrane androgen
receptors has not been addressed in colon cancer.
1.3 Apoptosis
Apoptosis is a form of programmed cell death that plays important roles during
animal development, immune response, elimination of damaged cells, and
maintenance of tissue homeostasis. It is associated with a distinct set of
biochemical and physical changes involving the cytoplasm, nucleus and plasma
membrane. The name was first introduced by John Kerr [Kerr JFR, et al. 1972]
in 1972, refers to the morphological feature of formation of ‘‘apoptotic bodies’’
from a cell. Carl Vogt, however, first described the phenomenon more than 100
years earlier in 1842. Now it has become a major research area in the
biomedical sciences.
1.3.1. Morphological features of apoptosis
Apoptosis has a lot of stereotypical morphological changes: The first is the cell
shrink which shows deformation and looses contact to its neighboring cells. Its
chromatin condenses and marginates at the nuclear membrane, then the cell
membrane begins to show blebs and eventually these blebs separate from the
dying cell and form "apoptotic bodies". The apoptotic bodies are engulfed by
macrophages and thus are removed from the tissue without causing an
inflammatory response. The apoptotic cells also cease to maintain phospholipid
asymmetry in the cell membrane, and phosphotidylserine appears on the outer
leaflet. The mitochondrial outer membrane also undergoes changes that include
loss of its electrochemical gradient, and substances like cytochrome c leak into
the cytoplasm. Finally, adjacent cells or macrophages phagocytose apoptotic
bodies and the dying cell. Those morphological changes are consequences of
characteristic molecular and biochemical events which occur in an apoptotic
cell. Most of them are activated notably by proteolytic enzymes. It finally
mediates the cleavage of DNA into oligonucleosomal fragments, in at the same
time as the cleavage of a multitude of specific protein substrates which usually
determine the integrity and shape of the cytoplasm or organelles [Saraste, A;
Pulkki, K 2000]. Furthermore, apoptosis is in contrast to the necrotic mode of
cell-death. During necrosis, the cellular contents are released uncontrolled into
the cell's environment, which results in damage of surrounding cells and a
strong inflammatory response in the corresponding tissue.
1.3.2. Molecular mechanisms of apoptosis signaling
pathways
Apoptosis is executed by intracellular proteases named caspases that are
activated during the onset of apoptosis via extrinsic and intrinsic pathways. The
intrinsic pathway is triggered by the release of proteins such as cytochrome c
from mitochondria to cytosol and the extrinsic pathway is activated by the
binding of death-inducing cytokines such as Tumor Necrosis Factor to its
receptor at cell surface. Both pathways are regulated at multiple steps to ensure
proper apoptosis.
The caspases are of central importance in the apoptotic signaling network
which is activated in most cases of apoptotic cell death [Bratton, SB, et al.
2000]. They belong to a group of enzymes known as cysteine proteases and
exist within the cell as inactive pro-forms or zymogens. These zymogens can be
cleaved to form active enzymes following the induction of apoptosis. Actually
cell death can only be classified to follow a classical apoptotic mode if execution
of cell death is dependent on caspase activity [Leist, M, Jaattela, M. 2001].
Caspases can be divided into three groups based upon structural differences
and substrate preferences, i.e. apoptotic initiators (caspase-2, -8, -9, and -10),
apoptotic executioners (caspase-3, -6, and -7), and cytokine processors
(caspase-1, -4, -5, -13, murine caspase-11, -12, and -14).
The extrinsic pathway is initiated by ligation of transmembrane death receptors
(Fas, TNF receptor, and TRAIL receptor) with their respective ligands (FasL,
TNF, and TRAIL) to activate membrane-proximal caspases (caspase-8 and –
10). It in turn cleaves and activates effector caspases such as caspase-3 and –
7. Typically the extrinsic pathway involves activating the initiator caspase,
caspase-8, which in turn either activates caspase-3 or cleaves the Bcl-2 family
member, Bid, leading to the formation of the apoptosome and activation of
caspase-9.
Besides amplifying and mediating extrinsic apoptotic pathways, mitochondria
also plays a central role in apoptosis. It regulates the integration and
propagation of death signals originating from inside the cell such as DNA
damage, oxidative stress, starvation, as well as those induced by
chemotherapeutic drugs [Kaufmann, SH, Earnshaw, WC. 2000; Wang, X. 2001].
This mitochondrial pathway named intrinsic pathways. The intrinsic pathway
requires disruption of the mitochondrial membrane and the release of
mitochondrial proteins. It involves members of the Bcl-2 family that regulate
cytochrome c released from the mitochondrial intermembrane space to
cytoplasm.
1.3.3. Apoptosis responses by AKT pathway
Akt is a good candidate for mediating PI3K-dependent cell-survival responses.
An important function of activated PI3K in cells is the inhibition of programmed
cell death [Yao and Cooper, 1995]. The first evidence to show that Akt acts as
an anti-apoptotic signaling molecule was observed in cerebellar granule
neurons after trophic factor withdrawal [Dudek et al., 1997], and in fibroblasts
after forced expression of c-Myc [Kauffmann-Zeh et al., 1997]. Subsequent
work in many laboratories has established the principle role of Akt in the
regulation of cell survival in several cell types, consistent with its ubiquitous
expression pattern. Akt has been implicated as an anti-apoptotic in many
different cell death paradigms, including withdrawal of extracellular signaling
factors, oxidative and osmotic stress, irradiation and treatment of cells with
chemotherapeutic drugs and ischemic shock [Franke et al., 1997; Downward,
1998]. Multiple studies supporting the role of Akt in apoptosis suppression have
connected Akt to cell death regulation either by demonstrating its
downregulation following pro-apoptotic insults, or by using gene-transfer
experiments that transduce both activated, anti-apoptotic and inactive, pro-
apoptotic mutants of Akt.
Taken together, these observations suggest that Akt may play a critical role both
in the function of cancer cells and in the pathogenesis of degenerative
diseases. By promoting the cell survival of mutated, damaged or transformed
cells even under adverse conditions, Akt can promote cancer cell growth by
protecting cells from apoptosis, which would otherwise be eliminated by
programmed cell death. To experimentally prove the importance of Akt kinases
in oncogenic transformation, in a seminal paper, Peter Vogt and colleagues
demonstrated that a transformed cellular phenotype could be reverted to normal
when using a cell model for PI3K-dependent oncogenesis as long as dominant-
negative mutants of Akt were expressed concomitantly [Aoki et al., 1998]. Akt is
also likely to play a significant role in degenerative diseases, where excessive
or inappropriate cell death occurs possibly because proper trophic factor
support is lacking. The relevance of Akt signaling in neurodegenerative disease
is supported by studies that examine its activity and function in Alzheimer's
disease models in vitro [Hong and Lee, 1997; Weihl et al., 1999]. A role for Akt
has also been suggested in other models of human degenerative diseases,
including cardiac failure [Matsui et al., 1999] and other cardiovascular diseases
where there is increased and chronic loss of cells [Reed and Paternostro,
1999].
1.3.4. Apoptosis responses by Bad
BAD is a distant member of the Bcl-2 family that promotes cell death.
Phosphorylation of BAD results in its cytosolic sequestration by the tau form of
14-3-3 proteins and its inactivation, as the phosphorylated form has reduced
ability to bind to membrane Bcl-xL. BAD phosphorylation induced by interleukin-
3 (IL-3) was inhibited by specific inhibitors of phosphoinositide 3-kinase (PI 3-
kinase) [Del Peso L, et al. 1997]. Although signals transduced by the
engagement of growth factor receptors, such as cytokines, insulin-like-growth
factor (IGF) and nerve growth factor (NGF), were long known to promote
survival, the molecular mechanisms linking their survival-promoting effect to the
direct inhibition of apoptosis emerged with the identification of select
components of the core apoptotic machinery that are modulated by
phosphorylation events downstream of survival signaling [Datta et al., 1999;
Amaravadi and Thompson, 2005]. BAD's capacity to bind and neutralize its anti-
apoptotic partners, BCL-2, BCL-XL and BCL-W, is inhibited on phosphorylation
by survival kinases activated by trophic factors. Various kinases have been
shown to phosphorylate BAD. S136 is a preferred substrate for AKT and p70S6
kinases in the PI3K signaling pathway [Datta et al., 1997; Del Peso et al., 1997;
Blume-Jensen et al., 1998; Eves et al., 1998; Harada et al., 2001]. S112 and
S155 harbor bona fide protein kinase A (PKA) consensus sites that can also be
recognized by p90 ribosomal S6 kinase (p90RSK), a kinase activated by the
MAPK pathway that shares multiple common substrates with PKA [Datta et al.,
2000; Tan et al., 2000; Houslay, 2006]. Modification of BAD by p90RSK is
consistent with several studies indicating that the activation of the
RAS/RAF/MEK/MAPK pathway modulates BAD phosphorylation [Bonni et al.,
1999; Fang et al., 1999; Scheid et al., 1999]. RAF can localize to mitochondria
[Wang et al., 1996; Gotz et al., 2005], and its activated forms promote BAD
phosphorylation [Fang et al., 1999]. However, RAF modulation of BAD
phosphorylation is likely indirect through other kinases such as AKT [Fang et al.,
1999; Wiese et al., 2001; von Gise et al., 2001; Gotz et al., 2005]. PIM kinases
constitute another class of survival kinases that phosphorylate BAD
predominantly on the S112 site [Fox et al., 2003; Yan et al., 2003; Macdonald et
al., 2006]. The interrelationship of these phosphorylation events is especially
intriguing as it suggests that BAD modification may serve as a node where
distinct signaling pathways converge to regulate the core apoptotic machinery.
Recent studies have proposed a sequential model of BAD dephosphorylation
initiated by pS112 dephosphorylation, which may then expose pS136 and
pS155 residues for dephosphorylation [Chiang et al., 2003]. Thus, both
phosphorylation and dephosphorylation of BAD at the three serine sites seem to
be tiered processes. Although S136 is the apical serine the phosphorylation of
which is needed for neutralizing BAD's apoptotic function, pS112
dephosphorylation may be the initial dephosphorylation event required for
promoting the apoptotic activity of BAD. It is also possible that, in addition to
directly targeting specific serine sites, BAD phosphatases may inactivate the
survival kinases [Andjelkovic et al., 1996; Djouder et al., 2007].
1.3.5. Apoptosis responses by actin polymerasion
Actin organization has been reported to trigger cell death and, potentially,
ageing. This function can lie upstream of mitochondrial ROS release [Gourlay
CW, Ayscough KR., 2005]. As it has been reported in yeast, in mammalian cells
actin stabilization can also induce cell death and this is mediated through
changes involving mitochondria [Posey, S. & Bierer, B. 1999, Odaka, C., et al.
2000]. A major regulator of actin that also has a role in apoptosis is gelsolin.
This protein is well known to mediate actin reorganization in response to
changes in Ca2+ and phosphoinositides in vivo and has a role in cell motility.
[Kwiatkowski, D. J. 1999] The role of gelsolin in regulating mitochondrial
potential might occur through a direct gelsolin binding to mitochondria. [Koya, R.
C. et al. 2000]. The key role of gelsolin to enhance actin depolymerization is in
protecting from apoptosis [Harms, C. et al. 2004]. The actin regulatory protein
cofilin has also been shown to have a key role in the apoptotic process by
promoting the depolymerization and severing of actin filaments. On the other
hand, changes in actin dynamics might trigger the activation of caspases.
In opossum kidney (OK) cells which express functional characteristics of normal
proximal tubular epithelial cells, TNF- has been reported exerts an apoptotic or
antiapoptotic cell response. And TNF- induces actin cytoskeleton
polymerization and their possible role to cell response in OK cells. The signaling
mechanism triggered by TNF- that leads to actin redistribution and to
modulation of NF- B and caspase-3 activity. The signal of TNF- elicits
antiapoptotic effects in OK cells is through the phosphatidylinositol-3 kinase (PI-
3 kinase) Cdc42 phospholipase (PLC)- 1 actin cytoskeleton polymerization
NF- B nuclear translocation cascade. [Papakonstanti EA, Stournaras C. 2004]
1.4 Cell Migration
Cells migrate in response to multiple situations they encounter during their lives.
In pathology, production of abnormal migratory signals may induce the
migration of the wrong cell type to the wrong place, which may have
catastrophic effects on tissue homeostasis and overall health. Some examples
include autoimmune syndromes in which immune cells home to certain
locations (joints in rheumatoid arthritis, and the CNS in multiple sclerosis are
two examples) and destroy the supporting tissue, causing severe damage; or
the process of metastasis, in which tumor cells abandon the primary tumor and
migrate to distant tissues where they generate secondary tumors.
There are different modes of cell migration depending on the cell type and the
context in which it is migrating. Cells can move as single entities, and the
specifics of their motility depend on several factors, adhesion strength and the
type of external migratory signals and cues, mechanical pliability,
dimensionality, and the organization of the cellular cytoskeleton. The intrinsic
properties of the cell interact with the environment to produce a migratory mode
or phenotype. Some tumor cells can move by extending membrane blebs, and
their actin cytoskeleton is not very organized, either. They have elaborate
cytoskeletal structures and adhesions, and their motion is generally slow. It is
worth noting that some cell types can switch between these depending on their
environment. Cells can also move in groups, including chains of cells and
sheet-like layers.
It is generally convenient to parse migration into a useful set of component
processes, which are often regulated by the same effectors regardless of the
cell type and the mode of migration. These processes include polarization,
protrusion and adhesion, translocation of the cell body and retraction of the rear.
These processes are coordinated and integrated by extensive transient,
signaling networks.
1.4.1. PI3K/Akt signaling pathways in cell migration
Migration is regulated by many gene products and complicated signaling
integrated in the concept of focalized adhesion. Main protagonists are protein
kinases such as extracellular signal-regulated protein kinase (ERK)1/2, which
are the most widely expressed members of the mitogen-activated protein (MAP)
kinase family, the phosphatidylinositol 3 kinase (PI3K), the focal adhesion
kinase (FAK) and others that can be activated by growth factors, cytokines and
ECM [Friedl and Wolf, 2003]. Interestingly, the importance of estrogens in
modulating rapid signaling effects that act on these targets has been recently
highlighted [Acconcia and Kumar, 2005; Acconcia et al, 2006]. A role for Akt in
the control of cell migration, invasion of the extracellular matrix, and ultimately
metastasis has been difficult to ascertain. Strikingly, activation of Akt1 has been
found to decrease mammary epithelial cell migration, and Akt1 prevents an
epithelial-to-mesenchymal transition that resembles events required for
metastasis [Irie et al., 2005] and [Yoeli-Lerner et al., 2005]. Two independent
mechanisms for this surprising Akt function have been explored. The first found
that the inhibitory effect of Akt1 on the in vitro migration and invasion properties
of breast cancer cell lines involved a pathway leading to degradation of the
nuclear factor of activated T cells (NFAT) transcription factors [Yoeli-Lerner et
al., 2005]. However, the molecular mechanism of Akt1-mediated degradation of
NFAT is currently unknown. A second group found that siRNA knockdown of
Akt1, but not Akt2, led to an increase in the migration of mammary epithelial
cells [Irie et al., 2005]. Loss of Akt1, specifically, led to an increase in the
activation of Erk1 and Erk2, which was found to be required for the enhanced
migration. Again, the mechanism by which Akt1, but not Akt2, inhibits Erk
signaling in this system remains unknown. Interestingly, mouse tumor models
have also suggested that Akt1 inhibits metastases [Hutchinson et al., 2004],
whereas Akt2 promotes metastases [Arboleda et al., 2003]. However, these
differential effects of Akt1 and Akt2 on epithelial cell migration may not translate
to other cell types. In fact, studies on cell migration using mouse embryonic
fibroblasts deficient of specific Akt isoforms have suggested opposite effects on
fibroblast migration, with Akt1 promoting migration and with Akt2 inhibiting it
[Zhou et al., 2006]. These studies demonstrate both the importance of crosstalk
between the PI3K-Akt pathway and other pathways and the emerging
recognition that the three isoforms of Akt can have distinct cellular functions.
1.4.2. Vinculin and cytoskeleton protein Actin in cell
migration
Vinculin is a ubiquitously expressed actin-binding protein. It used as a marker
for both cell–cell and cell–extracellular matrix (focal adhesion) adherens-type
junctions, but its function has remained elusive. A variety of phenotypes of
Vinculin-null cells have been shown that the role for vinculin include cell
adhesion, cell spreading, focal adhesion stability and strengthening, cell
migration and resistance to apoptosis. Vinculin regulate the focal adhesion
dynamics, and that transient increases in local phosphoinositide levels. This
effect inhibits the vinculin–F-actin interaction, promote focal adhesion turnover
and cell motility. Interestingly, the muscle-specific splice variant of vinculin called
metavinculin (which contains a 68 amino acid insert in the Vt domain), is
localized in dense plaques and costameres, cell–extracellular matrix junctions
that are much longer lived than focal adhesions. It could be significant that the
Vt/D5 domain of metavinculin interacts less strongly with acidic phospholipids
than does the Vt/D5 domain of vinculin [S. Witt et al., 2004]. The association of
metavinculin–vinculin heterodimers with F-actin might therefore be relatively
resistant to phospholipid competition. That association shows more persistent
adhesions. There is much recent interest in the finding that the globular head
region of vinculin (Vh) can participate in an intramolecular interaction with the
extended vinculin tail (Vt). This interaction masks the binding sites for talin and
α-actinin in Vh, F-actin in Vt, and VASP in the proline-rich region between Vh
and Vt. All this data has been reported that the cryptic actin-binding site in the
vinculin tail is exposed by PIP2. Interestingly, a talin-related peptide is shown to
change the vinculin head–tail interaction, unmasking the actin-binding site. [P.A.
Steimle, et al. 1999]. The paxillin-binding site in Vt is apparently not subject to
such regulation [A.P. Gilmore and K. Burridge. 1996]. The interaction between
Vh and Vt is relieved by acidic phospholipids [P.A. Steimle, et al. 1999, A.P.
Gilmore and K. Burridge. 1996, J. Weekes, et al.1996 and S. Huttelmaier, et
al.1998], which bind to Vt [R.P. Johnson, et al.1998], exposing the talin-, α-
actinin and VASP-binding sites and a cryptic protein kinase C (PKC)
phosphorylation site [J. Weekes, et al.1996, J. Weekes, et al. 1996] in Vt.
Whether the actin-binding site(s) in Vt is similarly exposed by acidic
phospholipids, as proposed originally [A.P. Gilmore and K. Burridge. 1996 and
J. Weekes, et al.1996], is contestable, and some study provides strong
evidence that PIP2 inhibits the interaction of Vt with F-actin in vitro [P.A.
Steimle, et al. 1999].
Vinculin can be tyrosine phosphorylated and some of the phosphorylation is Src
kinases dependent. [Zhiyong Zhang, et al. 2004] The tyrosine phosphorylation
sites in vinculin were reported to residues 100, 822and 1065. Tyrosine residue
1065 was phosphorylated by c-Src in vitro, but residue 100 was not
phosphorylated by c-Src in vitro, raising the possibility that the phosphorylation
of vinculin may be regulated by two, or more, distinct kinases. [Zhiyong Zhang,
et al. 2004] Following phosphorylation, vinculin tail showed significantly less
binding to the vinculin head domain than the unphosphorylated tail. Some
studies have shown that vinculin tail domain modulates the interaction between
paxillin and FAK, highlighting one mechanism by which a change in the tail
conformation may affect cellular responses. [Subauste MC, et al. 2004] Vinculin
phosphorylation residue 822 has been reported to affect interactions with
paxillin, which may be a valuable route to change cell motility and survival.
Actin polymerization and adhesion formation are linked. Actin polymerization
determines the rate of adhesion assembly and potentially nucleates adhesions
that contain activated integrins; conversely, adhesions provide nucleation points
that may support actin polymerization. Adhesions and actin are also physically
linked and this linkage coordinates adhesion assembly and disassembly and
the processes they regulate. Adhesion assembly requires actin polymerization
suggesting that the interaction of a subset of adhesion components with actin
nucleates the nascent adhesion, which is then stabilized by its association with
integrins. The direct interaction of focal adhesion kinase (FAK) and vinculin with
the Arp2/3 complex [DeMali et al., 2002; Serrels et al., 2007], the main
nucleator of actin branching and polymerization in lamellipodia, constitutes a
possible mechanism for targeting vinculin and FAK to future adhesion sites. The
presence of activated integrins in regions of protrusion outside adhesions
suggests that they enter the forming adhesion in an activated state [Galbraith et
al., 2007; Kiosses et al., 2001]. The other implication is that adhesions might
nucleate actin polymerization. This would provide a mechanism for the
formation of actin filaments on which adhesions elongate; these appear to
elongate from nascent adhesions at the lamellipodium-lamellum interface. This
possibility is supported by the observation that purified integrin-adhesion
complexes have actin-polymerization activity [Butler et al., 2006]. Although the
neutralization of Arp2/3 in β3-integrin-containing adhesion complexes did not
impair actin polymerization, targeting of the formin mDia did [Butler et al., 2006].
The organization and dynamics of the actin cytoskeleton are regulated by
membrane phosphoinositides at several levels. First, many actin-binding
proteins directly interact with phosphoinositides, which regulate the activity
and/or subcellular localization of these proteins. Among different PIs, PIP2 is the
best-characterized regulator of the actin cytoskeleton. PIP2 interacts directly
with several actin-binding proteins and regulates their activities [Hilpela P, et
al.2004, Sechi AS, Wehland J. 2000, Sheetz MP, et al. 2006, Yamaguchi H, et
al. 2009]. Typically, PIP2 inhibits those actin-binding proteins that promote actin
filament disassembly and activates proteins that induce actin filament assembly.
Second, phosphoinositides control the subcellular localization of larger
scaffolding proteins that are involved in the interplay between the actin
cytoskeleton and plasma membrane or intracellular membrane organelles.
Finally, proteins controlling the activity of Rho family small GTPases are in
many cases regulated by plasma membrane phosphoinositides. The RhoA
GTPase has a pronounced role in the formation and regulation of focal
adhesion complexes and contractile actomyosin bundles such as stress fibers
[Pelham RJ, et al. 1994]. RhoA induces actin polymerization at focal adhesions
by activating the Dia1 formin and inhibits actin filament disassembly by initiating
a signaling cascade that leads to phosphorylation and subsequent inactivation
of the ADF/cofilin family of actin filament severing/depolymerizing proteins
through the action of LIM kinases [Hotulainen P, Lappalainen P. 2006, Mahaffy
RE, Pollard TD. 2008, Watanabe N, et al. 1999, Vardouli et al 2005].
Furthermore, RhoA promotes contractility by activating the myosin light-chain
kinase through ROCK kinase [Totsukawa G, et al. 2000].
Focal complexes are regulated by signaling via Rac1 or cdc42 small GTPases
and are marked by the early recruitment of vinculin [J.V. Small et al. 2002 and
C.D. Nobes and A. Hall. 1995]. Vinculin is a large protein that contains binding
domains for multiple cytoskeletal proteins, including actin, α-actinin, talin,
paxillin, VASP, ponsin, vinexin and protein kinase C (PKC) [D.R. Critchley. 2000
and B. Geiger et al. 2001]. Its head and tail regions physically interact in a
resting state to mask most binding sites [D.R. Critchley. 2000]. The open,
‘activated’, conformation of vinculin is revealed by exposure to PIP2 and
exposes all binding sites. Past studies have revealed that vinculin plays a
central role in mechanical coupling of integrins to the cytoskeleton, as well as in
the control of cytoskeletal mechanics, cell shape, and protrusion amplitude and
cell motility. Vinculin binding to the arp2/3 complex might be but one way that
the actin-nucleation machinery can be coupled to new sites of adhesion, and
testing this hypothesis now presents cell biologists from different fields with a
fascinating new challenge.
1.5 Cell and animal model of colorectal cancer
The Caco-2 cell line is an immortalized line of heterogeneous human epithelial
colorectal adenocarcinoma cells, developed by the Sloan-Kettering Institute for
Cancer Research through research conducted by Dr. Jorgen Fogh. It has been
extensively used over the last twenty years as a model of the intestinal barrier.
When cultured as a monolayer, Caco-2 cells differentiate to form tight junctions
between cells to serve as a model of paracellular movement of compounds
across the monolayer. In many respects, the Caco-2 cell monolayer mimics the
human intestinal epithelium. [Richard B van Breemen , Yongmei Li. 2005]
Like Caco2 cell, HCT116 is another kind of human epithelial colorectal
adenocarcinoma cells. It established from the primary colon carcinoma of an
adult man; cells were described to carry a RAS mutation in codon 13 and to be
tumorgenic in nude mice.
IEC06 cells are derived from the rat small intestine and were produced from a
single clone. They were originally described by Quaroni and colleagues as a
homogenous population of epithelial-like cells. It has been shown with large,
oval nuclei, growing as tight colonies of polygonal, closely opposed cells
[Quaroni et al., 1979].
BALB/c mouse is an albino, laboratory-bred strain of the House Mouse. BALB/c
mice are useful for research into both cancer and immunology. They are
reported as having a low mammary tumor incidence. However, they can
develop other types of cancers, most commonly reticular neoplasms, lung
tumors, renal tumors and colon cancer.
ApcMin (Min, multiple intestinal neoplasias) is a point mutation in the murine
homolog of the APC gene. APC is tumor suppressor gene, which involved in
causing colorectal cancer. It is involved in both sporadic and familial forms of
colorectal cancer. A mutation in the APC gene is the earliest detectable
molecular abnormality in colorectal cancer. APCMin/+ mouse model carries
defective APC, which results in the spontaneous development of colorectal
tumors.
37
2. Aims of the studies
From the studies published so far, it became evident that functional mARs
trigger potent antitumor effects in prostate and breast cancer cells. These
findings imply a potential role of mAR as a novel target for the development of
selective cancer treatments (reviewed in Papadopoulou et al., 2009).
However, it remained elusive whether mARs are also expressed in other
tumors and whether their stimulation could result in the induction of anti-tumor
effects similar to the ones described in prostate and breast cancer cells. The
aim of the present work was to study the expression of functional mARs in
colon cancer tissue. The main goal of this work was to address the role of
mAR activation toward major characteristics of tumor cells, namely cell
survival and cell migration. Because mAR activation has been shown to
promote strong apoptotic regression (Kampa M, et al.2002, Gu S, et al.2009),
we analyzed the functionality of the prosurvival PI-3K/Akt pathway. In addition,
because this signaling pathway also plays a major role in the invasive
potential of tumor cells, we further studied the migration potential upon mAR
activation. Our findings indicate that prosurvival signaling prevails in colon
tumor cells but is strongly downregulated upon mAR stimulation in vitro and in
colon tumor tissues isolated from BALB/c and APCMin/+ mice following
treatment with mAR agonists. Furthermore, mAR activation blocked migration
and invasiveness of colon tumor cells, mainly recruiting the adhesion-and
actin cytoskeleton–regulator vinculin. This work provides novel mechanistic
insights into the regulation of the proapoptotic and antimigratory mAR effects
in colon tumors.
38
3. Materials and methods
3.1 Materials
3.1.1. Chemical and biological reagents
1,2-dimethylhydrazine (DMH; Sigma-Aldrich, St.Louis, USA)
Annexin V-FITC (BD Biosciences,USA)
anti-actin antibody (Cell signaling, Beverly, US)
anti-tubulin antibody (Cell signaling, Beverly, US)
ApoAlert® Caspase Colorimetric Assay kit (Clontech, USA)
APOPercentage Apoptosis Assay kit (Biocolor,Carrickfergus, UK)
aprotinin (Sigma-Aldrich, St. Louis, US)
APS (Merck, Darmstadt, Germany)
Biomax x-flim (Kodak, Rochester, US)
BSA (Roth, Karlsruhe, Germany)
Developing solution (Kodak, Rochester, US)
Dihydrotestosterone (DHT) (Sigma-Aldrich, St. Louis, US)
DMEM medium (Gibco, Carlsbad, US)
DRAQ-5 dye (Biostatus, Leicestershire, UK)
ECL detection reagent (Amersham, Louisville, UK)
EGTA (Sigma-Aldrich, St. Louis, US)
estradiol (Sigma-Aldrich, St. Louis, US)
fetal bovine serum (Gibco, Carlsbad, US)
filter paper (Whatman, Maidstone, UK)
FITC-conjugated goat anti-rabbit IgG(Molecular Probes, Eugene, US)
fixing solution (Kodak, Rochester, US)
formaldehyde (Roth, Karlsruhe, Germany)
goat anti rabbit IgG antibody (Amersham, Louisville, UK)
goat serum (Invitrogen, Carlsbad, US)
gold antifade reagent (Invitrogen, Carlsbad, US)
HSA-FITC(Sigma-Aldrich, St. Louis, US)
in situ Cell Death Detection Kit, Fluorescein ( Roche, Basel, Switzerland )
39
methanol (Roth, Karlsruhe, Germany)
NaCl (Roth, Karlsruhe, Germany)
NaF (Sigma-Aldrich, St. Louis, US)
nonfat milk (Roth, Karlsruhe, Germany)
Paraformaldehyde (Roth, Karlsruhe, Germany)
PBS tablet (Gibco, Carlsbad, US)
PCA (Sigma-Aldrich, St. Louis, US)
phalloidin (Sigma-Aldrich, St. Louis, US)
PMSF (Sigma-Aldrich, St. Louis, US)
PVDF blot membrane (Millipore, Billerica, US)
rhodamine-phalloidin (Molecular Probes, Eugene, OR)
SDS loading buffer (Roth, Karlsruhe, Germany)
sodium citrate (Roth, Karlsruhe, Germany)
sodium orthovanadate (Roth, Karlsruhe, Germany)
standard pelleted food (C1310, Altromin, Heidenau, Germany)
sucrose (Sigma-Aldrich, St. Louis, US)
synthetic dextran sulfate sodium (DSS; Wako Pure Chemical Industries, Led.
Japan)
TEMED (Roth, Karlsruhe, Germany)
testosterone-HSA ((Sigma-Aldrich, St. Louis, US))
Testosterone-HSA-FITC(Sigma-Aldrich, St. Louis, US)
Tris (Roth, Karlsruhe, Germany)
Tris-HCl (Roth, Karlsruhe, Germany)
Triton X-100 (Roth, Karlsruhe, Germany)
TWEEN-20 (Roth, Karlsruhe, Germany)
40
3.1.2. Equipment
-20°C refrigerator (Liebherr, Lindau, Germany)
4°C refrigerator (Heraeus, Massachusets, US)
-80°C refrigerator (Sanyo, Osaka, Japan)
Balence (Sartorius, Goetingen, Germany)
Biorad ChemiDoc XRS (Biorad, Hercules, US)
Cell culture hood (Thermo, Waltham, US)
Cell incubator (Heraeus, Massachusets, US)
Centrifuge 22R (Heraeus, Waltham, US)
Centrifuge 5417 R (Eppendorf, Hamburg, Germany)
Confocal Laser Scanning Microscope (Carl Zeiss, Jena, Germany)
Cyrostat (Thermo, Waltham, US)
Electrophoresis cell (Biorad, Hercules, US)
Electrophoresis power supply (Biorad, Hercules, US)
Electrophoretic transfer cell (Biorad, Hercules, US)
Folie bag sealer (Roth, Karlsruhe, Germany)
Heator (Schutron, Pocklington, UK)
Magnetic stirrer (Roth, Karlsruhe, Germany)
Pippets (Abimed, Langenfeld, Germany)
Shaker (Roth, Karlsruhe, Germany)
Spectronic GENESYS 6 UV-Vis Spectrophotometer (Thermo, Waltham, US)
Vortex (Peqlab,Erlangen, Germany)
Waterbath (Labortechnik, Seelbach, Germany)
41
3.2 Methods
3.2.1. Cell culture
The Caco2 human colon cancer cell lines and IEC06 non transformed
intestinal cells were obtained from the American Type Culture Collection
(Manassas, VA) and were studied between passages 60 and 70. CACO2 at
20,000/ml were cultured in DEME medium supplemented with 20% fetal
bovine serum in culture flasks in a CO2 incubator at 37°C. Based on previous
titration experiments [Gu et al., 2009] we have used throughout this study a
10-7 M testosterone-HAS concentration for mAR stimulation.
3.2.2. Preparation of steroid solution
Before each experiment testosterone-3- (O-carboxymethyl) oxime-Human
Serum Albumin, referred to as testosterone-HSA (or Testo-HSA), DHT and
estradiol, were dissolved in serum-free culture medium at a final concentration
of 10-5 M. This stock solution was incubated for 30 min at room temperature
with 0.3% charcoal and 0.03% dextran, centrifuged at 3000 x g and passed
through a 0.45 μm filter to remove any potential contamination with free
steroid. Testosterone-HSA, estradiol and DHT solutions were used at a final
concentration of 10-7 M throughout all studies. If not otherwise stated all
treatments and incubations with steroids including apoptosis assays were
performed in serum-containing medium. Testosterone-HSA-FITC or control
HSA-FITC constructs were generated by conjugating Testosterone-HSA or
HSA with FITC using standard techniques.
3.2.3. In vivo animal experiment
Colon carcinoma was generated as described previously (Wang et al., 2004).
In a first series of experiments, 7-week old Balb/c mice (both male and
female) were divided into two groups, A (n=5) and B (n=7). Both groups
42
underwent carcinogenic treatment. At the age of 9 weeks animals were
subjected to three cycles of alternating administration of distilled water
containing 30 g/L synthetic dextran sulfate sodium (DSS; molecular mass
5000 Da; Wako Pure Chemical Industries, Led. Japan) for 7 days followed by
distilled water for subsequent 14 days after intraperitoneal pretreatment with
20 mg/kg 1, 2-dimethylhydrazine (DMH; Sigma-Aldrich Corp.
St.Louis.MO.USA). Group B mice received in addition to the carcinogenic
treatment 5 mg/kg testosterone-HSA subcutaneously injected three times per
week throughout the study period. All mice were sacrificed at the age of 20
weeks. After death, the entire colorectum from the colorectal junction to the
anal verge was examined. Fresh specimens were placed in liquid nitrogen
and subsequently stored at -80°C for further analysis. Then, the colon was
opened longitudinally, washed with PBS, and divided into three portions
(proximal, middle and distal). After macroscopic inspection the colon was fixed
in a 40% g/L formaldehyde buffer solution (pH.7.4).
In APC mice, animal experiments were carried out in mice of either sex with
mutated apc resulting in spontaneous colon tumor development (APCMin/+)
obtained from the Jackson Laboratory (USA). The animals were housed under
controlled environmental conditions (22-24°C, 50-70% humidity and a 12-h
light/dark cycle). Throughout the study the mice had free access to standard
pelleted food (C1000, Altromin, Lage, Germany) and tap water. All animal
experiments were conducted according to the German law for the care and
welfare of animals and were approved by local authorities.
The animals were divided into two groups. Group A, (n = 6) received
treatment of 5 mg/kg subcutaneous TAC injection three times per week for 8
weeks. In the control group B (n=4) similar doses of normal saline were given.
At the end of 8 weeks all animals were anesthetized with ether and sacrificed.
After death, the entire colorectum from the colorectal junction to the anal
verge was examined. Then, the colon was opened longitudinally, washed with
PBS, and divided into three portions (proximal, middle and distal). Tumors
were counted with a dissecting microscope at x3 magnification. After
inspection the colon was fixed in a 40% g/L formaldehyde buffer solution
43
(pH.7.4).
3.2.4. Immunofluorescence analysis and confocal laser
scanning microscopy
For testosterone-HSA-FITC staining, 5-µm-thick frozen tissue sections from
the Balb/c or APC mouse tumors were fixed with 4% PFA for 15 min and
incubated with 5% BSA/1x PBS/0.3% Triton for 1 hour at room temperature.
After two washes with PBS 1.5% FBS specimens were exposed to
testosterone-HSA-FITC (10-7 M, Sigma) for 1h at room temperature. Nuclei
were stained with DRAQ-5 dye (1:1000, Biostatus, Leicestershire, UK) for 10
min at room temperature.
For direct fluorescence microscopy of F-actin, cells were fixed with 3 %
paraformaldehyde in PBS for 30 min, permeabilized with 0.5 % Triton X-100 in
PBS (10 min) and incubated with rhodamine-phalloidin (Molecular Probes,
Eugene, OR, 1:100 dilution) for 40 min in the dark. For indirect
immunofluorescence staining, cells were incubated for 2h at room
temperature with mouse monoclonal anti-tubulin (Cell signaling, 1: 1000
dilution). Secondary FITC-conjugated rabbit anti-mouse IgG (Invitrogen) was
used in a 1: 200 dilution. Nuclei were stained with DRAQ5™ (Biostatus
Limited). Slides were mounted using the ProLang® Gold Antifade reagent
(Invitrogen).
To quantify the expression of phosphorylated Akt and Bad, 5-µm-thick frozen
tissue sections from the APC mice colon tumors were fixed with 4% PFA for
15 min at room temperature. After washing twice with PBS the slides were
incubated with 5% normal goat serum/1x PBS/0.3% Triton for 1 hour at room
temperature. Then, the specimens were exposed overnight at 4°C to
phospho-Akt (Thr308) (1:800, Cell Signaling, USA) or phospho-Bad (Ser136)
(1:100, Santa Cruz Biotechnology, CA). The slides were rinsed three times
with PBS and incubated for 1.5 h at room temperature with secondary FITC
goat anti-rabbit antibody (1:500, Invitrogen, UK). After three washing steps the
44
nuclei were stained for 10 min at room temperature with DRAQ-5 dye (1:1000,
Biostatus, Leicestershire, UK).
To determine the phosphorylation of Vinculin, cells were cultured on glass
cover slips with testosterone-HSA or control without testosterone-HSA for
different time periods indicated in the figure legends. After washing twice with
PBS, cells were incubated with 4% PFA for 15 min and then incubated with
5% normal goat serum/1x PBS/0.3% Triton for 1 hour at room temperature.
Then, the cells were exposed to anti-vinculin antibodies (1:400, Gene Tex,
USA) at 4°C overnight. The cells were rinsed three times with PBS and
incubated with secondary FITC goat anti-rabbit antibody (1:500, Invitrogen,
UK) or goat anti-mouse antibody (1:500, Invitrogen, UK) for 1.5 h at room
temperature. For F-actin staining, cells were incubated with rhodamine-
phalloidin (1:100, Molecular Probes, Eugene, OR) for 40 min in the dark. After
three washing steps the nuclei were stained with DRAQ-5 dye (1:1000,
Biostatus, Leicestershire, UK) for 10 min at room temperature. All the slides
and coverslips were mounted with ProLong Gold antifade reagent
(Invitrogen).
All Images were taken on a Zeiss LSM 5 EXCITER Confocal Laser Scanning
Microscope (Carl Zeiss MicroImaging GmbH, Germany) with a water
immersion Plan-Neofluar 40_/1.3 NA DIC. Images were analyzed with the
instrument's software.
3.2.5. Immunoprecipitation and Westen blotting
Cells were incubated with 10-7 M testosterone-HSA for the indicated time
periods, washed twice with ice cold PBS and suspended in 500 μl ice-cold
lysis buffer (50mM Tris/HCl, 1% TritonX-100 pH 7.4, 1% sodium deoxycholate,
0.1% SDS, 0.15% NaCl, 1 mM EDTA, 1 mM sodium orthovanadate)
containing protease inhibitor cocktail (Sigma). The protein concentration was
determined using the Bradford assay (BioRed). Sixty µg of protein were
solubilized in sample buffer at 95ºC for 5 min and resolved by 10% SDS-
PAGE. For immunoblotting proteins were electro-transferred onto a PVDF
45
membrane and blocked with 5% nonfat milk in TBS-0.10% Tween 20 at room
temperature for 1 h. Then, the membrane was incubated with phospho-Akt
(Thr308), phospho-PI-3K p85 (Tyr458)/p55 (Tyr199) (1:1000, Cell Signaling,
USA), or phospho-Bad (Ser136) (1:100, Santa Cruz Biotechnology, CA) at
4°C overnight. After washing (PBST) and subsequent blocking the blot was
incubated with secondary anti rabbit antibody (1:2000, Cell Signaling, USA) or
anti-mouse antibody (1:5000, GE Healthy, USA) for 1 h at room temperature.
After washing, antibody binding was detected with the ECL detection reagent
(Amersham, Germany). For controls the blots were stripped in stripping buffer
(Carl Roth, Germany) at 56°C for 30 min. After washing with PBST blots were
blocked with TBST + 5% milk for 1 h at room temperature. Then, they were
incubated with anti-Akt, or anti-Bad (1:100, Santa Cruz Biotechnology, CA)
antibodies at 4°C overnight. After washing with PBST and incubation with anti-
rabbit antibody (1:2000, Cell Signaling, USA), antibody-binding was detected
and quantified with Quantity One Software (Biorad, Germany).
For immunoprecipitation, equal amounts of protein (500 μg) in the presence or
absence of 50μM genistein were subjected to immunoprecipitation with a
monoclonal anti-phosphotyrosine antibody (7 μg/500 μg total protein, Santa
Cruz Biotechnology, CA). After incubation with the antibody for 1h at 4°C, 50μl
of the homogeneous protein A suspension was added into the mixture and
incubated overnight at 4°C on a rocking platform. After three washing steps,
samples were resuspended in SDS sample buffer, subjected to SDS
electrophoresis and transferred to nitrocellulose membrane. Proteins were
incubated with vinculin monoclonal antibody (1:100, Santa Cruz
Biotechnology, CA) followed by the appropriate anti-mouse antibody (1:5000,
GE Healthy, USA). Detection of protein bands was succeeded with ECL kit.
Bands were quantified with Quantity One Software (Biorad, Germany).
3.2.6. Measurement of G/total actin ratio by Triton X-100
fractionation
For measurements of the monomeric (Triton soluble) and polymerized (Triton
46
insoluble) actin, Caco2 cells were incubated for different time point with or
without or testosterone-BSA (10.7 M). Then, 500 μl of Triton-extraction buffer
(0.3% TritonX-100; 5 mM Tris, pH 7.4; 2 mM EGTA; 300 mM sucrose; 2 μM
phalloidin; 1 mM PMSF; 10 μg/ml leupeptin; 20 μg/ml aprotinin; 1 mM sodium
orthovanadate; and 50 mM NaF) were added, and the mixture was incubated
for 5 min on ice. After removing the buffer, soluble proteins were precipitated
with equal volumes of 6% PCA. The Triton-insoluble fraction remaining on the
plate was precipitated with 1 ml of 3% PCA. Equal volumes of each fraction
were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
The resulting protein-bands were transferred onto nitrocellulose membrane,
and the membrane was blocked with 5% nonfat dry milk in TBS-T (20 mM
Tris, pH 7.6; 137 mM NaCl; 0.05% Tween-20) for 1 h at room temperature.
Antibody solutions (in TBS-T) were added for 1 h at room temperature rabbit
anti-actin first antibody and second horseradish peroxidase-coupled antibody
(Chemicon, Temecula, CA)]. Blots were developed by using the ECL system,
and the band intensities were quantitated by PC-based image analysis.
3.2.7. Matrigel and transwell assay
Matrigel assays were performed using BD BioCoat™ BD MatrigelTM Invasion
Chambers (BD Bioscience, USA). Matrigel was placed in each insert with 8.0
μm pore size in a 24-well plate. The chamber was allowed to polymerize at
37°C for 1 h. The inserts were then washed with serum-free DMEM. 100 μl of
complete cell culture medium with 1 × 105cells was then seeded onto the
insert. Five hundred μl of complete cell culture medium with 10-7 M
testosterone-HSA or testosterone-BSA (Sigma) in the presence or absence of
10-6 M cytochalasin B (Sigma) was added into the well below the insert. In
control experiments cells were pre-incubated with 10-7 M testosterone
albumin conjugates for 2 h. Then, TAC was washed out with complete cell
culture medium and five hundred μl were added into the well below the insert.
After a 24 h incubation, the insert was wiped with a wet cotton swab. The
lower surface was gently rinsed with PBS, the cells were fixed and stained
with DAPI for 10 minutes, rinsed again with sterile water and allowed to dry.
After removing the membranes from the inserts, they were mounted with
47
ProLong Gold antifade reagent (Invitrogen). To determine the total number of
migrating cells, the slices were viewed and imaged under the microscope, and
the number of cells/field in 10 random fields was counted. Experiments were
performed in triplicates.
The transwell assay was performed using transwell inserts (BD Bioscience,
USA). The inserts were then washed with serum-free DMEM, 100 μl of
complete cell culture medium with 1 × 105 cells were seeded onto the insert.
500 μl of complete cell culture medium with 10-7 M testosterone-HSA or
testosterone-BSA (Sigma) in the presence or absence of 10-6 M cytochalasin
B (Sigma) 10-6 M anastrozole (Sigma), 10-6 M flutamide (Sigma), 5*10-6 M
PP2 (Sigma), or 5*10-6 M genistein (Sigma) was added into the well
underside of the insert for 24 h at 37°C and 5% CO2. In control experiments
cells were pre-incubated with 10-7 M testosterone albumin conjugates for 2 h.
Then, TAC was washed out with complete cell culture medium and five
hundred μl were added into the well below the insert. After 24 h incubation,
cells were fixed, stained with DAPI for 10 min and microscopically analyzed
as described above.
3.2.8. Wound healing assay
For the wound healing assay, confluent cells cultures were scraped with a
pipette tip across a 24-well-plate. Following wounding, culture medium was
replaced with fresh medium and cells were exposed to 10-7 M testosterone-
HSA (Sigma) in the presence or absence of 10-6 M cytochalasin B (Sigma) for
the indicated time points. It should be noted that the methodology for the
wound healing assay requires that confluent cells are used, in order to provide
the necessary cell layer to create wounding by scraping. On the other hand,
subconfluent cells were used in matrigel and transwell assays (see below), to
ensure correct evaluation of cell motility and invasiveness.
3.2.9. siRNA experiments
Caco2 cells were grown in DMEM medium containing 10% fetal calf serum
48
under standard culture conditions (37°C, 5% CO2). 4x104 cells were seeded
in 24 well plates and cultivated with fresh culture medium for 8 h. The cells
were subsequently transfected with validated siRNA for Vinculin (ID# s14764,
Ambion, Darmstadt, Germany) or with a negative control siRNA using siPORT
Amine (Ambion) transfection agent according to the manufacturer’s protocol.
The efficiency of silencing was checked by Western blot 72 h after
transfection. Upon silencing, 37.6% of the vinculin protein was still detectable
in cells treated with siRNA for Vinculin compared to cells treated with a
negative control siRNA.
3.2.10. TUNEL assay
The colonic cancer tissue was cut to 8 μm frozen sections and subsequently
fixed in 4 % paraformaldehyde for 30 min at room temperature. After rinsing
with PBS the samples were permeabilized in a solution of 0.1 % Triton X-100
in sodium citrate for 2 min. Samples, washed with PBS, were then incubated
in the TUNEL reaction mix for 1 h at 37oC, according to the manufacturer’s
instructions (Roche, Germany). Nuclei were stained with DRAQ5™ (Biostatus
Limited). Sections were analyzed with a confocal laser scanning microscope
(Carl Zeiss).
3.2.11. APOPercentage apoptosis assay
Caco2 cells were cultured in 96-well plates for the APOPercentage apoptosis
assay (Biocolor Ltd., Belfast, Ireland). In the presence or absence of 10-6 M
anastrozole (Sigma), they were stimulated or not with 10-6 M TAC for 24
hours in serum containing medium. Untreated cells cultured in serum free
medium were used as positive control for the apoptotic response.30 mins
before the incubation time is reached, add 5 μl APOPercentage Dye to the
centre of the well. Incubate for the remaining 30 min of the assay. Then
Syringe off the culture medium/dye mixture, and gently wash the cells twice
with 200 μl/well PBS. For apoptosis quantitation, the amount of dye within the
labeled cells can subsequently be released into solution, and the
concentration is measured at a wavelength of 550 nm, using Spectronic
49
GENESYS 6 UV-Vis Spectrophotometer (Thermo, Waltham, US).
3.2.12. Statistical analysis
Data are provided as means ± SEM; n represents the number of independent
experiments. Data were tested for significance using unpaired student’s t-test
when two-sample means were tested. Differences were considered
statistically significant when p-values were < 0.05. All statistical analysis was
performed with GraphPad InStat version 3.00 for Windows 95, GraphPad
Software, San Diego California USA, www.graphpad.com.
50
4. RESULTS
4.1 mAR expression in colon cancer cell lines
In my diplom thisis, while analyzing in vivo mAR expression in paraffin blocks
generated from xenograft tumor tissues of various origins, we have noticed
significant mAR expression in colon cancer xenograft specimens. In line with
these findings mAR expression was subsequently detected by confocal laser
scanning microscopy using the fluorescent testosterone-HSA-FITC conjugate
in cultured HCT116- (Figure5 e,f), or in Caco2-colon cells (Figure 5a,b) while
HSA-FITC labeled CaCo2 or HCT116 cells showed no apparent staining
(Figures 5 c, d, g, h). These results indicate that mAR in expressed in colon
cancer cell lines. Interestingly, mAR staining could not be detected in the non-
transformed intestinal cell line IEC06 (Fig. 6A). These staining experiments
and the fact that testosterone-HSA-FITC is an impermeable conjugate
disclosed mAR expression preferentially in colon cancer cell lines and tumors.
In addition, mAR could be also detected in iAR silenced of Caco2 cells by
using testosterone-HSA-FITC. These results imply that the molecular identity
of mAR is probably not identical with iAR (Fig. 6B)
Figure 5 Membrane staining of mAR in Caco2 and HCT 116 colon cancer cells
51
Confocal laser scanning microscopic analysis of Caco2 cells (a-d) and HCT 116 cells(e-h)
stained with testosterone-HSA-FITC, showing specific FITC related fluorescence at the cell
membranes, or HSA-FITC, showing no apparent membrane staining. Visualization of nuclei
was evident by DRAQ5™ or TO-PRO-3 staining. Magnification, ×100.
A
B
Figure 6 Membrane staining of mAR in IEC 06 cells and iAR silenced Caco2 cells.
A) Confocal laser scanning microscopic analysis of IEC 06 cells (a-b) stained with
testosterone-HSA-FITC, showing no apparent membrane fluorescence at the cell membrane.
Visualization of nuclei was evident by DRAQ5™ staining.
B) Confocal laser scanning microscopic analysis of iAR silenced cells (a-d) stained with
testosterone-HSA-FITC, showing membrane fluorescence at the cell membrane. Visualization
of nuclei was evident by DRAQ5™ staining.
52
4.2 mAR expression in 2 different colon cancer animal
models
The findings provided so far indicate that mAR are expressed in colon cancer
cell lines Caco2 and HCT-116 in vitro. [Gu S, et al. 2009] Thus, we aimed to
further evaluate the in vivo effects of albumin-conjugated androgens in colon
cancer animal models. To this end we first estimated the expression of mAR
in colon tumors generated in Balb/c mice and APC mice. As shown in figure 7,
using testosterone-HSA-FITC we detected specific, FITC-related fluorescence
in membrane specimens of Balb/c mice colon tumors (Fig. 7 A, a, a1), and
APC mice colon tumors (Fig.8). No apparent staining could be identified in
tissues labeled with HSA-FITC (Fig. 7A, b) and no apparent staining could be
found in healthy tissue labeled with testosterone-HSA-FITC (Fig. 7B).
Figure 7: In vivo testosterone-HSA expression in BALB/c mice
A) Confocal laser scanning microscopic analysis of BALB/c colon tumor frozen sections
stained with testosterone-HSA-FITC (a, a’), showing specific FITC related fluorescence at the
cell membranes. No apparent membrane fluorescence was shown in control samples stained
with HSA-FITC (b).
B) Confocal laser scanning microscopic analysis of BALB/c colon tumor and healthy frozen
sections stained with testosterone-HSA-FITC, showing specific FITC related fluorescence at
the cell membranes of tumor sections.
53
Figure 8: In vivo testosterone-HSA expression in APC mice
Confocal laser scanning microscopic analysis of APCMin/+ colon tumor frozen sections
stained with testosterone-HSA-FITC, showing specific FITC-related fluorescence at the cell
membranes. Visualization of nuclei was evident by DRAQ5™ staining. Magnification, ×100.
4.3 mAR activation by testosterone-HSA was followed
by extensive reduction of tumor incidence in vivo
Having a clear indication for mAR-expression, the 12-week tumor incidence of
colon tumors generated in Balb/c mice was assessed by chemical
carcinogenesis (see Experimental Procedures) in the presence or absence of
continuous testosterone-HSA treatment. The animals used for these studies
were divided in two groups comprising 5 and 7 animals. One group (7
animals) was treated subcutaneously (3 times/week, for 12 weeks) with
5mg/kg testosterone-HSA, whereas the other group (5 animals) remained
untreated. The results (Figure 9, A) show that testosterone-HSA-treatment
produced a clear and significant reduction of tumor incidence by 65%. The
histological analysis of tumors by Tunel assay (Figure 9, B) confirmed that
apoptotic cells were present in significant numbers predominantly at the
tumors of animals treated with testosterone-HSA, while they were significantly
less in the non-treated animals. These results collectively show that mAR is a
functional target that may be used for the selective elimination of colon cancer
cells in vivo.
54
Figure 9: In vivo testosterone-HSA effects on tumor incidence in BALB/c mice
A) Arithmetic means ± SEM of colonic tumor incidence in BALB/c mice. Following treatment
with the carcinogenic drug 1,2 dimethylhydrozine followed by dextrane sodium sulphate, one
group (7 animals) was treated subcutaneously (3 times/week for 12 weeks) with 5mg/kg
testosterone-HAS (black bar), whereas the other group (5 animals) remained untreated (white
bar). # indicates significant difference between both groups (# P<0.01).
B) After treatment, the colonic cancer tissue was cut to 8 μm frozen sections and fragmented
DNA was assessed using TUNEL assay according to the manufacturer's instructions.
Confocal laser scanning microscopy analyzed samples. Magnification, ×100.
To further establish the in vivo role of mAR activation in mice model, in a
second series of experiments APC mice have been used. In these
experiments, animals were divided in two groups comprising 6 and 4 animals.
One group (6 animals) was treated subcutaneously (3 times/week, for 8
weeks) with 5mg/kg testosterone-HSA, whereas the other group (4 animals)
remained untreated. As shown in Fig. 10A, testosterone-HSA treatment
resulted in a significant reduction of the tumor incidence by 80%. The
histological analysis of tumors by TUNEL assay confirmed that apoptotic cells
were present in appreciable numbers predominantly in the tumors of animals
treated with testosterone-HSA (Fig. 10B, left panels) whereas they were
significantly less abundant in the non-treated animals (Fig. 10B, right panels).
55
Figure 10: In vivo testosterone-HSA effects on tumor incidence in APCMin/+
mice
A) Arithmetic means ± SEM of colonic tumor incidence in APC Min/+
mice. The mice were either
treated with 5 mg/kg testosterone-HSA subcutaneously 3 times/week for 8 weeks, (n=6
animals black bar) or treated with 5 mg/kg of normal saline subcutaneously 3 times/week for 8
weeks (n=4 animals white bar). # indicates significant difference between both groups (**
P<0.01).
B) After treatment, the APCMin/+
colonic cancer tissue was cut to 8 μm frozen sections, and
fragmented DNA was assessed using TUNEL assay according to the manufacturer's
instructions. Confocal laser scanning microscopy analyzed samples. Magnification, ×100.
4.4 p-Akt and p-Bad are downregulated in colon tumor
tissues treated by testosterone-HSA
Activated Akt is a pro-survival factor controlling phosphorylation and activity of
various pro-apoptotic gene products [ Cardone MH, et al. 1998, Datta SR, et
al. 1997]. Moreover, the role of this kinase in inactivating the pro-apoptotic
function of Bad via phosphorylation is well-documented [Downward J. 1998,
Vanhaesebroeck B, et al. 2000]. Finally, downregulation of Akt and Bad has
been reported previously in mAR-stimulated prostate cancer cells
(Papadopoulou et al Mol Canc 2008) implying that this prosurvival signaling
may be down regulated by mAR. Accordingly, the activity of AKT and Bad was
further analyzed in colon tumor tissues treated or not with testosterone-HSA.
To address this, first I assessed the 8-week incidence of colon tumors
56
spontaneously developed in APCMin/+ mice in the presence or absence of
continuous testosterone-HSA treatment. In our experiments, animals were
divided in two groups comprising 6 and 4 animals. One group (6 animals) was
treated subcutaneously (3 times/week, for 8 weeks) with 5mg/kg testosterone-
HSA, whereas the other group (4 animals) remained untreated.
Immunohistochemical analysis with either anti p-Akt (Thr308) or anti p-Bad
(Ser136) antibodies revealed strong expression of p-Akt and p-Bad in colonic
tumor tissue from untreated mice and marked downregulation of both
phosphorylated proteins in colon tumor tissues from testosterone-HSA treated
animals (Fig. 11). These results clearly prove the anti-tumor potential of
testosterone-albumin conjugates in colon cancer and corroborate previously
reported data obtained in a chemically induced colon tumor model in Balb/c
mice. Furthermore, our results identify Akt/Bad as downstream targets of mAR
in colon cancer in vivo.
Figure 11: In vivo testosterone-HSA stimulation inhibits Akt activity and induces Bad
de-phosphorylation in APCMin/+ mice
Confocal laser scanning microscopic analysis of TAC-treated and untreated APCMin/+ frozen
colon tumor sections stained with (a) anti-phospho-Akt (Thr308) and (b) anti-phospho-Bad
57
(Ser136) antibodies. Anti-rabbit-FITC was used as secondary antibody and DRAQ5™ for
nuclei staining. Magnification, ×100.
4.5 mAR stimulation inhibits Akt activity and induces
Bad de-phosphorylation in Caco2 but not in IEC06
cells
Having established a role of AKT/Bad downstream of mAR activation in colon
cancer tissues, we sought to determine the possible inactivation of p-Akt /p-
Bad in colon cancer cells treated by testosterone-HSA. Consistent with its well
documented oncogenic role in tumor cells, high basal levels of active,
phosphorylated Akt (p-Akt) were detected in Caco2 colon tumor cells (Fig
12A). Stimulation of mARs with testosterone-albumin conjugates (TAC)
induced a long-term and profound de-phosphorylation of this kinase that
became evident 2 hours upon TAC treatment and sustained for at least 12
hours (Fig. 12A). Interestingly, in non transformed IEC06 intestinal cells that
do not express mAR [ Gu S, et al. 2009], p-Akt levels were very low and
remained unchanged during TAC treatment (Figure 10C), indicating mAR
specificity for the regulation of this pro-survival factor. Moreover, Figure 1B
clearly shows that mAR stimulation by TAC resulted in de-
phosphorylation/activation of Bad following kinetics similar to that of Akt,
reaching minimum levels of phosphorylated Bad (p-Bad) after 12 h (Fig. 12B).
In line with these findings, the Akt-upstream regulator PI-3K was de-
phosphorylated upon long term TAC treatment (Fig 12D), implying that the
pro-survival PI-3K/Akt signaling is downregulated in mAR activated Caco2
cells.
58
Figure 12: De-phosphorylation/inactivation of Akt and Bad in Caco2 and IEC 06 cells.
A) Caco2 cells were exposed to 10-7
M testosterone-HSA for the indicated time periods. The
ratio of the cellular content of the phosphorylated residues (Thr 308) versus the total isoform
of Akt was measured in cell lysates by Western blotting using specific antibodies for each
form and was normalized to the corresponding control.
B) Caco2 cells were exposed to 10-7
M testosterone-HSA for the indicated time periods. The
ratio of the cellular content of the phosphorylated residues (Ser 136) versus the total isoform
of Bad was measured in cell lysates by Western blotting using specific antibodies for each
form and was normalized to the corresponding control.
C) IEC 06 cells were exposed to 10-7
M testosterone-HSA for the indicated time periods. The
ratio of the cellular content of the phosphorylated residues (Thr 308) versus the total isoform
of Akt was measured in cell lysates by Western blotting using specific antibodies for each
form and was normalized to the corresponding control.
D) Caco2 cells were exposed to 10-7
M testosterone-HSA for the indicated time periods. The
59
ratio of the cellular content of the phosphylated PI3K versus the total isoformof PI3K was
measured in cell lysates by Western blotting using specific antibodies for each form and was
normalized to the corresponding control.
Blots show a representative experiment, the numbers below each lane correspond to the
mean values ± SE from three independent experiments (*P<0.05; **P<0.01***P<0.001)
indicating the fold-decrease in the phosphorylation level for the indicated time point
normalized to the controls.
4.6 mAR activation triggered rapid actin and tubulin
reorganization in colon cancer cells
Cytoskeleton reorganization is a prominent early functional response of
various cancer cells to steroid hormones targeting membrane binding sites
[Koukouritaki et al., 1997, Kampa et al., 2002, Kampa et al., 2006,
Papadopoulou et al., 2008a]. Accordingly, to analyze the functional impact of
mAR in colon cancer rapid cytoskeleton modifications was investigated in
Caco2 cells upon activation of mAR with testosterone-HSA for various time
intervals. Cellular actin cytoskeleton dynamics were initially assessed by
appropriate quantitative techniques as described in Papakonstanti et al.,
2007. As shown in figure 13A, quantitative immunoblot analysis of Triton X-
100 insoluble cytoskeletal pellets and corresponding supernatants revealed a
significant decrease of the Triton-soluble (monomeric) to total actin ratio in
Caco2 cells treated with 10-7 M testosterone-HAS, indicating actin
polymerization. This effect was evident 15 min upon testosterone-HSA
treatment; and returned to nearly control levels after 1-2 hours (Fig. 13A). The
quantitative data were fully supported by confocal laser scanning microscopic
analysis, showing redistribution of microfilamentous structures and formation
of stress fibers and filopodia in testosterone-HSA treated cells (Fig. 13B).
60
Figure 13: Modulation of the dynamic equilibrium G- and Total actin in testosterone-
HSA stimulated Caco2 cells.
24h serum starved cells were stimulated with 10 -7
M androgen conjugate for the indicated time points. A) Total and G- actin were measured by quantitative immunoblot analysis after Triton X-100
subcellular fractionation. Bars present the G/Total actin mean value SE of four independent duplicate experiments (** P < 0.01). B) Cells were stained with rhodamine-phalloidin for filamentous actin and DRAQ5™ for nuclei.
Tubulin cytoskeleton reorganization was further analyzed by confocal laser
scanning microscopy. A clear redistribution of the microtubular network
became evident in cells treated with 10-7 M testosterone-HSA for 15 to 60
minutes (Fig. 14).
61
Figure 14: Modulation of the dynamic equilibrium rapid tubulin reorganization in
testosterone-HSA stimulated Caco2 cells.
Caco2 cells treated or not with 10
-7 M testosterone-HSA for different time points were cultured
in coverslips, fixed and stained with rabbit anti-α-tubulin. Anti-rabbit-FITC was used as
secondary antibody and DRAQ5™ for nuclei staining. Confocal laser scanning microscopy
analyzed samples. Magnification, ×100.
Previously, it has been reported that activation of mAR with non permeable
testosterone derivatives induced pro-apoptotic responses [Gu S Diploma
Thesis, Gu S, et al. 2009]. However, the mechanism regulating the mAR-
induced apoptotic responces is still unknown. In recent years, the cross-talk
between actin cytoskeleton components and apoptotic signaling has attracted
specific interest. Indeed, modifications of actin dynamics seem to be crucial
for apoptotic responses [Gourlay CW, et al. 2005, Franklin-Tong VE,et al.
2008]. More recently the functional role of actin reorganization in regulating
the pro-apoptotic responses induced by mAR was established in prostate
cancer cells [Papadopoulou N, et al. 2008,] Based on these results we
assessed the mAR-dependent apoptosis and caspase-3 activation in the
presence of anti-actin drugs. As shown in Figures 15A,B, in Caco2 cells pre-
62
treated with cytochalasin B, at a concentration (10-7M) which blocks actin
redistribution without exerting toxic effects [Stournaras et al 1996], the mAR-
induced apoptotic response (Fig 15A) and caspase-3 activation (Fig 15B)
were abolished. These results indicate that actin redistribution is a mandatory
step for the apoptotic response of mAR-stimulated colon cancer cells.
TAC: Testosterone-HSA Cyto B: cytochalasin B
Figure 15: Pro-apoptotic effects of testosterone-HSA, DHT and Estradiol in the absence
or presence of inhibitors in Caco2 cells.
A) Quantitative APOPercentage apoptosis assay of testosterone-HSA stimulated Caco2 cells
and similar experiments in the presence of cytochalasin B (Cyto B). Cells were exposed to 10-
7 M testosterone-HSA for 24 hours and proapoptoric responses were assessed by the
APOPercentage apoptosis assay. Equally, cells pre-treated or not with 10-7
M Cyto B or
flutamide, were exposed to testosterone-HSA for 24 hours. Cells serum starved for
comparable periods of time served as a positive control for apoptosis. Bars present the mean
OD measured at 550 nm. ** P<0, 01, n=4.
B) Cells were pre-treated or not with Cyto B for 1h and then exposed or not to 10-7
M
testosterone-HSA for 4h, lysed and incubated with the caspase-3 substrate DEVD conjugated
to the chromophore pNA according to the manufacturer's instructions. Caspase-3 activity was
measured at 405 nm. ** P<0, 01, n=4.
63
4.6 mAR activation inhibits cell motility in colon
cancer cells
We further examined whether mAR-stimulation regulates motility and
invasiveness of colon cancer cells. To this end, we assessed the migration
capacity of Caco2 cells treated or not with testosterone-HSA by using the
matrigel-, transwell- and wound healing-assays, respectively. As shown in
Figure 16, the matrigel assay revealed that cell invasiveness was reduced by
90% following testosterone-HSA-treatment of Caco2 cells. Very similar results
were also obtained when cells were treated with testosterone-BSA, indicating
that both testosterone albumin conjugates produce similar effects in the
inhibition of cell motility. In line with this, cell motility assessed by the wound-
healing assay confirmed the inhibition of the migratory capacity of colon
cancer cells by mAR stimulation (Fig. 17A). The inhibition of the migratory
potential of tumor cells by mAR stimulation was further corroborated by the
transwell assay (Fig. 17B). Interestingly, in the presence of non toxic
concentrations of cytochalasin B that block the well described mAR-induced
actin reorganization the inhibition of cell motility was partially restored in all
experimental procedures (Fig 16, 17).
Figure 16: Effect of testosterone-HSA on Caco2 human colon cancer cell invasion
Cells were cultured with 10-7
M testosterone-HSA in the presence or absence of 10-6 M
cytochalasin B on the Matrigel-coated upper compartment of Transwell culture chambers,
64
provided with an 8 μm pore size polycarbonate filter, according to the manufacturer’s
instructions. 24 h later, Matrigel was removed by scraping, and invaded cells, attached to the
lower surface of the filter, were stained with DAPI. The slices were imaged under the
microscope and the number of cells in 10 random fields was counted. Bars represent the % of
cell invasion in control and treated cells. (n=3).
Figure 17: Effect of testosterone-HSA on Caco2 human colon cancer cell migration
A) Wound healing assay of Caco2 cells. Following 24 h culture the confluent monolayer was
scratched with a pipette tip to create a cell-free area. Testosterone-HSA 10-7
M was added, and
wound closure was documented by microphotography of the same region after 24 h. Bars
represent the width of the wound in control and treated cells.
B) Cells in the presence or not of 50μM genistein and PP2 were cultured with 10-7 M
65
testosterone-HSA in the absence or presence of 10-6 M cytochalasin B on the Transwell
culture chambers, provided with an 8 μm pore size polycarbonate filter, according to the
manufacturer’s instructions. 24 hours later, invaded cells, attached to the lower surface of the
filter, were stained with DAPI. The slices were imaged under the microscope, and the number
of cells in 10 random fields was counted. Bars represent the % of cell invasion in control and
treated cells. (n=3), *P<0,05; ***P<0,001
Finally, to further confirm the androgen specificity of the mAR induced effects,
we analyzed cell motility and the apoptotic response of TAC treated Caco2
cells in the presence of the aromatase inhibitor anastrozole. As shown in
Figure 18 and 19, both, the migration potential and the apoptotic response
could not be influenced by this inhibitor.
Figure 18: Motility effects of testosterone-HSA in the absence or presence of inhibitors
in Caco2 cells
Cells in the presence or not of 10-6 M anastrozole or 10-6 M flutamide were cultured with 10-7 M
testosterone-HSA on the Transwell culture chambers, provided with an 8 μm pore size
polycarbonate filter, according to the manufacturer’s instructions. 24 hours later, invaded cells,
attached to the lower surface of the filter, were stained with DAPI. The slices were imaged under
the microscope, and the number of cells in 10 random fields was counted. Bars represent the
% of cell invasion in control and treated cells.
66
Figure 19: Pro-apoptotic effects of testosterone-HSA in the absence or presence of
inhibitors in Caco2 cells
Quantitative APOPercentage apoptosis assay of TAC stimulated Caco2 cells and similar
experiments in the presence of anastrozole. Cells were exposed or not to 10-7
M
testosterone-HSA for 24 hours and proapoptoric responses were assessed by the
APOPercentage apoptosis assay. Equally, cells pre-treated or not with 10-6
M anastrozole
was exposed to testosterone-HSA for 24 hours. Cells serum starved for comparable periods
of time served as a positive control for apoptosis. Bars present the mean OD measured at
550 nm. *** P<0,001, n=3.
4.7 mAR activation triggers vinculin phosphorylation
The findings presented in Figure 16 and 17 suggested that actin restructuring
may control cell migration upon mAR activation in colon cancer cells. As
tyrosine phosphorylation of specific proteins is linked to actin cytoskeleton
dynamics and cellular motility [ Wozniak MA, et al. 2009, Zhao J, et al. 2009],
we sought to determine whether mAR effects on cell motility were evident in
the presence of genistein and PP2 that represent widely used tyrosine
phosphorylation inhibitors. Fig. 17B shows that this was indeed the case, as
genistein and PP2 efficiently blocked mAR induced inhibition of cell migration.
Notably, both inhibitors had no effect on its own in this assay. We
subsequently checked for molecular targets potentially regulating these
67
effects. We focused on vinculin, an adhesion protein that participates in cell-
cell adhesions and that was described to regulate migration and actin
organization [ Bailly M, et al.2003]. As shown in Fig 20A, mAR stimulation with
testosterone-HSA disclosed a substantial increase in the phosphorylation of
vinculin within 15 min remaining at least for 12 h. Confocal microscopy
analysis fully confirmed the western blotting data. Indeed, immunostaining of
testosterone-HSA-treated Caco2 cell preparations with anti-vinculin antibodies
(Fig. 20B) revealed gradually increased vinculin spots observed at the focal
adhesions. Triple immunostaining with anti-vinculin antibodies, rhodamine-
phalloidin for F-actin staining and DRAQ-5 dye nuclear staining clearly
documented the formation of actin filaments emanating from the vinculin spots
of focal adhesions (Fig. 20B).
Figure 20: Effects of testosterone-HSA on vinculin phosphorylation and on Caco2 cell
morphology
A) Caco2 cells were stimulated with 10-7
M testosterone-HSA for the indicated time periods.
Following cell lysis equal amounts of proteins were immunoprecipitated (IP) with an anti-
phosphotyrosine (p Tyr) antibody. The tyrosine-phosphorylated as well as equal amounts of
68
total lysates were immunoblotted (IB) with a specific antibody against vinculin. The
immunoblots were analyzed by densitometry. The intensity of phosphorylated vinculin bands
was normalized to the intensity of the corresponding total vinculin bands. Blots show a
representative experiment, whereas the relative fold increase (mean values ± SE from three
independent experiments) in vinculin phosphorylation with that of untreated cells taken as 1
are indicated. (**P<0, 01)
B) Confocal laser scanning microscopic analysis of vinculin and actin in mAR- activated
Caco2 cells. Cells treated or not with 10-7
M testosterone-HSA for different time periods were
cultured on coverslips, fixed and stained with mouse anti-vinculin, anti-mouse-FITC as
secondary antibody, DRAQ5™ for nuclei staining and rhodamine-phalloidin for filamentous
actin staining. Magnification, ×100.
Interestingly, in the presence of the tyrosine phosphorylation inhibitor
genistein, vinculin phosphorylation was effectively blocked (Figure 21A).
Moreover, confocal laser scanning analysis revealed that both the expression
of the characteristic vinculin spots and the formation of actin filaments, shown
in Fig. 20B, disappeared in mAR-stimulated Caco2 cells in the presence of
genistein (Fig. 21B). These results, coupled with the observed blocking effect
of genistein in mAR-induced cell motility of CaCo2 cells (Fig. 17B), provide
indirect evidence that vinculin phosphorylation may participate in mAR
signaling towards actin reorganization and cell motility regulation.
69
Figure 21: Phosphorylation of Vinculin in TAC-treated Caco2 cells in the presence of
genistein
A) Caco2 cells in the presence of genistein were stimulated with 10-7
M Testosterone-HSA for
the indicated time periods. Following cell lysis equal amounts of proteins were
immunoprecipitated (IP) with an anti-phosphotyrosine (p-Tyr) antibody. The tyrosine-
phosphorylated as well as equal amounts of total lysates were immunoblotted (IB) with a
specific antibody against the vinculin. The intensity of phosphorylated vinculin bands was
normalized to the intensity of the corresponding total vinculin bands.
B) Confocal laser scanning microscopic analysis of vinculin and actin in mAR-activated
Caco2 cells in the presence of genistein. Cells treated with 10-7
M testosterone-HSA for
different time periods were cultured in coverslips, fixed and stained with mouse anti-vinculin,
anti-mouse-FITC as secondary antibody, DRAQ5™ for nuclei staining and rhodamine-
phalloidin for filamentous actin staining. Magnification, ×100.
4.8 Vinculin is necessary for actin reorganization and
migration of mAR stimulated Caco2 cells
To provide direct and specific experimental evidence for the regulatory action
of vinculin in mAR-stimulated Caco2 cells, we designed short interfering
70
RNAs (siRNAs) that could induce down-regulation of endogenous vinculin.
The siRNA was capable of inducing knockdown of endogenous vinculin to
almost 70% of the control levels when introduced into Caco2 cells by transient
transfection (Fig. 22A). The effect of the siRNA was specific, as an unrelated
siRNA had no apparent effect on the protein levels of vinculin (Fig. 22A).
Analysis of vinculin and actin cytoskeleton of cells under such conditions of
siRNA transient transfection showed a dramatic inhibitory effect on the ability
of testosterone-HSA to affect vinculin morphology and actin reorganization
(Fig. 22B). In contrast, the control siRNA did not interfere with the actin and
vinculin responses to testosterone-HSA.
Figure 22: Effect of testosterone-HSA on Caco2 cells silenced with vinculin siRNA
A) Caco2 cells transfected with vinculin siRNA or a negative control siRNA were lysed, and
71
equal amounts of total lysates were immunoblotted (IB) with a specific antibody against
vinculin and GAPDH. The immunoblots were analyzed by densitometry.
B) Confocal laser scanning microscopic analysis of vinculin and actin in mAR-activated
Caco2 cells silenced either with vinculin siRNA or a negative control siRNA. Transfected cells
treated with 10-7
M testosterone-HSA for different time periods were cultured in coverslips,
fixed and stained with mouse anti-vinculin, anti-mouse-FITC as secondary antibody,
DRAQ5™ for nuclei staining and rhodamine-phalloidin for filamentous actin. Magnification,
×100.
Vinculin was shown to be important in regulating adhesion dynamics and cell
migration [Huynh N, et al. 2010], and it was postulated that vinculin may
connect early adhesion sites to the actin-driven protrusive machinery [ Zhao J,
et al. 2009]. In addition, the relative decrease of endogenous vinculin levels
led to a significant restoration of the migratory capacity of Caco2 cells upon
mAR activation, as indicated by both the matrigel- and transwell-assays (Fig
23).
72
Figure 23: Effect of testosterone-HSA on Caco2 cells silenced with vinculin siRNA
A) Representative motility experiment of Caco2 cells transfected or not with vinculin siRNA.
Cells were cultured in the presence of 10-7
M testosterone-HSA on the Matrigel-coated upper
compartment or Transwell culture chambers, provided with an 8 μm pore size polycarbonate
filter, according to the manufacturer’s instructions. 24 h later, invaded cells, attached to the
lower surface of the filter, were stained with DAPI.
b) Quantification of the motility experiments shown in (C) for Caco2 cells silenced with
vinculin siRNA and cultured in the presence of 10-7
M testosterone-HSA as described in Fig.
14 and 15 B.
Similar results were obtained in cells pre-treated with testosterone-HSA for 2h
prior assessing the migration potential by the matrigel- and transwell-assays
in the presence or absence of siRNAs against vinculin or the corresponding
73
negative siRNA control (Fig 24). This experiment also suggests that the
observed inhibitory signals on cell migration and adhesion upon testosterone-
HSA treatment are triggered very early upon mAR activation when cells are
still viable and cannot be attributed to an artificial response of dying cells
measured after 24h of TAC treatment (as presented in Fig. 16 and 17). We
conclude that vinculin is a critical component downstream of mAR that
regulates the responses to actin cytoskeleton reorganization and migration
potential in colon tumor cells.
Figure 24: Effect of testosterone-HSA on Caco2 cells silenced with vinculin siRNA Quantification of control matrigel and transwell motility experiments with Caco2 cells pre-
treated with 10-7
M testosterone-HSA for 2 h and silenced with either negative control siRNA
or vinculin siRNA. Bars represent the % of cell invasion in control and treated cells. (n=3),
***P<0.001.
74
5. Discussion
In recent years a lot of studies introduced the concept of nongenomic steroid
hormone actions, which explains the observations related to rapid steroid
effects. Nongenomic steroid actions have been reported for most prominent
steroids [Losel R, Wehling M 2003, Heinlein CA, and Chang C 2002].
Although the nature of these membrane steroid sites was elusive until recently,
the identification of a membrane progesterone receptor [Zhu Y, et al 2003,
Falkenstein E, et al 1999, Falkenstein E, et al 1996, Zhu Y, et al 2003] and the
isolation of a membrane glucocorticoid-binding protein with homologies with -
opioid receptors [Evans SJ, et al 2000] show that these proteins might belong
to the seven-transmembrane G protein-coupled receptors. This hypothesis is
further supported by recent findings showing that the orphan G protein-
receptor, GPRC6A can be activated by anabolic steroids, including
testosterone. This report suggests that GPRC6A may mediate the non-
genomic effects of testosterone and other anabolic steroids [Pi M,et al 2010 ].
Colorectal cancer is one of the most common cancers and the third leading
cause of cancer death. The existence of sex differences in colon cancer
incidence was proposed several years ago. The neoplasia occurs more often
in men than in women in nearly all countries [Haenszel, W. and Correa, P.
1971]. The colon cancer has been reported to be related to a lot of
intracellular steroid receptors, like intracellular estrogen receptor, intracellular
thyroid hormone receptor and intracellular androgen receptors [I. D_Errico
and A. Moschetta 2008]. The importance of intracellular androgen receptors in
colorectal cancer is supported by many avenues of research. They prove that
androgen receptors show altered binding characteristics in colon cancer.
However, It still remained unknown whether mARs are also expressed in
colon cancer and whether their activation could result in the induction of anti-
tumorigenic effects similar to those described in other cancer cells.
In the present work we provide experimental evidence that membrane
androgen receptors are expressed in colon tumors. Using tissue specimens
from colon tumors and established colon tumor cell lines we show here that
colon cancer cells express functional mARs. [Gu S, et al. 2009] Moreover,
75
membrane-impermeable testosterone albumin conjugates induce
considerable apoptosis via activation of the pro-apoptotic executor caspase-3.
[Gu S, et al. 2009] The observed mAR-activated effects are specific and
independent from the classical intracellular androgen receptors (iAR), since
they were manifested in the presence of the anti-androgen flutamide. In
addition, mAR staining could be also detected in iAR silenced Caco2 cells
(Fig 6B) and iAR-deficient DU145 human prostate cancer cells [Hatzoglou A,
et al. 2005]. All those imply that the molecular identity of mAR is probably not
identical with iAR, targeted to the plasma membrane.
5.1 Membrane androgen receptor activation in colon cancer
triggers pro-apoptotic responses in vitro and in vivo
The results from my diplom thesis have show that membrane-impermeable
testosterone albumin conjugates induced considerable apoptosis via
activation of the pro-apoptotic executor caspase-3. Moreover, the results from
other studies indicated as well that membrane androgen receptors are
predominantly expressed in tumor cells. Activation of these receptors triggers
pro-apoptotic responses. One possible rationalization for the expression of
those receptors is that tumor cells may compensate mAR-dependent
apoptosis by over-expressing anti-apoptotic proteins or other compensatory
mechanisms that collectively protect against mAR-dependent pro-apoptotic
effects. Previous reports support this assumption: Indeed, iAR deficient
DU145 human prostate cancer cells were shown to over-express the pro-
survival PI-3K/Akt pathway, which was down-regulated following long-term
mAR activation [Papadopoulou N, et al. 2008A]. In addition, the FAK/PI3K
pathway was constitutively activated in DU145 cells and mAR activation was
unable to further alter the short-term phosphorylation levels of those kinases
[Papadopoulou N, et al. 2008], while long term activation induced significant
de-phosphorylation [Papadopoulou N, et al. 2008A]. In these cells, PI-3K was
constitutively activated [Papadopoulou N, et al. 2008], whereas long-term
mAR stimulation by specific agonists induced dephosphorylation of both, PI-
3K and its downstream effector Akt [Papadopoulou N, et al. 2008A].
76
In this study, pro-survival signals are effectively downregulated in mAR-
expressing colon tumors following stimulation by testosterone-albumin
conjugates (TAC) both, in vitro and in vivo. This was concluded by findings
showing that phosphorylated kinase Akt, which is constitutively upregulated in
colon tumors but not in non-transformed cells (Fig 12 A, C), was significantly
downregulated upon long-term mAR activation by TAC. In line with this, PI-3K
was de-phosphorylated upon long term TAC treatment (Fig 12 D), implying
that the pro-survival PI-3K/Akt signaling is downregulated in mAR activated
Caco2 cells. In addition, the pro-apoptotic Bad protein was efficiently
dephosphorylated and thus activated by TAC following similar kinetics (Fig.
12B). These results suggest that, in contrast to non transformed intestinal
IEC06 cells, mAR-expressing colon tumor cells express activated pro-survival
signals that may protect them from apoptotic cell death. mAR activation
downregulated the activity of these signals via dephosphorylation, a finding
consistent with the strong apoptotic regression upon mAR stimulation
reported recently [Gu S, et al. 2009].
Recent studies using mouse xenografts have shown that a testosterone–
albumin conjugate (testosterone-BSA) induced potent apoptotic regression of
prostate tumors in vivo [Hatzoglou A, et al. 2005]. In addition, testosterone-
BSA was also reported to potentiate the paclitaxel-mediated cytotoxicity both
in vitro and in vivo [Kampa M, et al. 2006]. These reports are supported by in
vivo experimental findings presented in this work. Indeed, when Balb/c mice
were treated with testosterone-HSA and the 12-week tumor incidence of colon
tumors was assessed the chemically induced tumors were reduced by 65% in
the testosterone-HSA-treated animals. Most probably this effect was due to
the apoptotic regression of tumor cells as indicated by the Tunel assay. These
results point out clearly that activation of mAR by testosterone-HSA
significantly affects the incidence of colon tumors in vivo and they are in line
with the previously reported prostate tumor regression in mice [Hatzoglou A,
et al. 2005, Kampa M, et al. 2006]. Interestingly, mAR is strongly expressed in
tissues derived from p53-deficient xenograft tumors. Since p53 is a frequently
inactivated gene in tumors, it is interesting to hypothesize that mAR activation
77
may result in eradication of p53 tumors in vivo. Notwithstanding the above,
and despite the fact that additional experiments are required for the detailed
evaluation of mAR-dependent biological effects in colon cancer, our data
support the recently postulated notion [Papadopoulou N, et al. 2009] that
mAR may represent a novel specific tumor target.
These results were also confirmed by in vivo experiments in APCMin/+ mice.
This mouse model is carrying defective adenomatous polyposis coli (APC)
resulting in gastrointestinal tumors being developed spontaneously [ Gourlay
CW, et al. 2005]. Indeed, the colon tumor incidence observed in the TAC-
treated animals was significantly reduced by 80% (Fig. 10A). Interestingly,
and in line with the findings in colon tumor cells pointing to downregulation of
pAKT/pBad (Figure 11) histological immunostaining analysis revealed that
both, p-Akt and p-Bad were effectively downregulated in colon tumor
specimens isolated from TAC-treated animals (Fig. 13). These findings
collectively provide novel mechanistic evidence, pointing to p-Akt/p-Bad
signaling, which may control the mAR-induced anti-tumorigenic effects in vitro
and in vivo. It should be noted that APCMin/+ mutant mice of either sex were
used in this study to avoid conflicting data regarding the sex-related
prevalence of colon tumors of APCMin/+ mutant mice. It is pointed out that the
enhanced susceptibility of male mice to intestinal tumor growth [Smith KJ, et
al. 1993, Hinoi T, et al 2007] results rather from the classical androgen
receptor, since mAR anti-tumorigenic effects seem to be independent from the
classical androgen receptor signaling [Hatzoglou A, et al. 2005, Gu S, et al.
2009, Papakonstanti EA, et al. 2003, Papadopoulou N, et al. 2008].
Taken together, the results presented in prostate and colon tumors imply that
mAR expression is associated with active pro-survival pathways thus
protecting cells from apoptotic regression. Activation of these receptors
triggers specific signaling to restrict this pro-survival machinery. This
assumption is fully supported by the in vitro and in vivo results presented
here, arguing that the in vitro findings reported previously are not simply a
side effect of mAR activation.
78
5.2 Membrane androgen receptor activation blocks
migration
The connection between actin cytoskeleton components and androgen
signaling has attracted specific interest in recent years [Ting HJ, et al. 2008].
Actin dynamics seem to be crucial for apoptotic responses [Gourlay CW, et
al.2005, Franklin-Tong VE, et al.2008]. The findings in our present work
further underscored the key role of actin cytoskeleton rearrangements in
regulating apoptosis. Indeed, it was clearly shown that actin (and tubulin)
reorganization represent major early events following mAR activation by
testosterone-HSA. Moreover, early blockade of actin rearrangement by
depolymerizing drugs e.g. cytochalasin B, virtually abrogated the pro-
apoptotic responses (Fig. 15A, B). The involvement of the early actin
rearrangement in mediating the late apoptotic responses was addressed in
earlier studies in prostate cancer cells. In these studies it was shown that
inhibition of either up-stream or down-stream signals regulating early actin
polymerization blocked the late activation of NFkB and FasL signaling
[Papadopoulou N, et al. 2008A]. Although this pro-apoptotic signaling was not
addressed in the present study we hypothesise that the actin reorganization is
an early functional step in the pro-apoptotic response. These findings, which
are in close agreement with similar results reported recently in prostate cancer
cells treated with testosterone albumin conjugates [Papadopoulou N, et al.
2008 and 2008A], further emphasize the functional cross-talk between
cytoskeleton rearrangements and regulation of apoptosis [Gourlay CW, et
al.2005, Franklin-Tong VE, et al.2008].
Akt has been also shown to play a major role in the invasive potential of colon
cancer cells in response to a variety of stimuli e.g. hergulin [Yoshioka T, et al.
2010], PAK1 [Huynh N, et al. 2010], Sprouty-2 [Holgren C, et al.2010].
Moreover, inhibition of Akt-dependent pathways has been linked to reduced
cell motility in colon cancer [Lai TY, et al. 2010], while mAR stimulation
downregulates p-Akt [Papadopoulou N, et al. 2008A and this work] and mAR-
dependent activation has been show to block cell motility and invasion on
79
prostate cancer cells [Hatzoglou A, et al. 2005]. Thus, we sought to determine
an effect of mAR activation on cell motility in colon tumor cells. The present
observations further reveal that mAR stimulation modulates specific molecular
targets controlling cell motility. Activation of mAR by two distinct testosterone
albumin conjugates (testosterone-HSA and testosterone-BSA) markedly
inhibited cell motility as documented by different assays (Fig 16 and 17). The
possibility that testosterone conjugates may be converted to estrogen and
influence the overall interpretation of our results was also considered.
However, previous binding studies in colon cancer [Gu S, et al. 2009] as well
as in prostate cancer cells [Kampa M, et al. 2002] have clearly indicated that
estrogens (and progesterone) displaced radiolabeled testosterone with
significant lower affinity (104 to 102-fold). These findings imply that even if
such a conversion takes place, it cannot influence the mAR induced effects
described so far, because of the high androgen selectivity of these membrane
receptors. In addition, control experiments showed that neither migration nor
apoptotic responses were influenced by the aromatase inhibitor anastrozole
(Figure 18 and 19), further supporting the androgen specificity of the mAR
induced effects. Finally, since flutamide did not influence the motility effects
nor the apoptotic responses in colon cancer [Gu S, et al. 2009], it is believed
that the mAR pool mediating the observed effects is most likely unrelated to a
membrane-associated form of the classical, intracellular AR that may be
present in the plasma membrane of colon tumor cells. Although the existence
of such form of membrane tethered intracellular AR has not been reported in
colon cells, experimental data in prostate cells place iAR on the cell
membrane [Lind GE, et al. 2004]. In that case, however, and in sharp contrast
to what we have observed in our assays in colon tumors, membrane
associated iAR induced cell proliferation (instead of apoptosis) which was
efficiently blocked by anti-androgens or anti-estrogens [Lind GE, et al. 2004].
In conclusion, our data support the existence of an active, non-AR/ER related
membrane receptor bearing anti-cancer action in colon cancer cells. This
conclusion is in line with recent findings demonstrating that iAR could not be
detected in membrane preparations of CaCo2 cells [Gu S, et al. 2009].
Inhibition of migration is usually correlated with impaired expression/activation
80
of adhesion molecules and reorganization of focal contact structures,
including actin cytoskeleton [ Yoshioka T, et al. 2010]. Polymerization of actin
filaments against cellular membranes provides the force for a number of
cellular processes such as migration, morphogenesis, and endocytosis
[Saarikangas J, et al.2010]. Since actin reorganization is a major effect of
mAR activation in tumor cells [Kampa M, et al. 2002, Kallergi G, et al. 2007,
Gu S, et al. 2009, Papadopoulou N, et al. 2008], we focused on the molecular
mechanism underlying the mAR-induced inhibition of cellular motility in Caco2
cells (Fig. 16 and 17). Our results demonstrate that vinculin is a main target of
mAR activation that may regulate cell motility. Phosphorylation of vinculin (Fig.
20A) was an early and persistent event leading to significant morphological
changes of Caco2 cells. It was clearly correlated with actin restructuring as
indicated by the visualization of newly organized actin filaments emanating
from the vinculin spots on the cell adhesion contacts (Fig. 20B). Interestingly,
vinculin silencing or inhibition of vinculin phosphorylation reversed largely
actin reorganization and the inhibition of migration. These findings imply that
vinculin phosphorylation/activaton upon mAR stimulation regulates cell
adhesion and inhibits the migration potential of Caco2 tumor cells. This
conclusion is in line with several reports in the literature. Thus, vinculin was
shown to be important in regulating adhesion dynamics and cell migration
[Huynh N, et al. 2010], and it was postulated that vinculin may connect early
adhesion sites to the actin-driven protrusive machinery [ Zhao J, et al. 2009].
According to this model vinculin stabilizes focal adhesions and thereby
suppresses cell migration, an effect that is relieved by modifications of inositol
phospholipids [Holgren C, et al.2010]. Although the precise role of vinculin in
focal adhesions remains to be elucidated, recent experimental evidence
suggest that vinculin overexpression reduces cell migration, whereas vinculin
downregulation enhances cell motility [Holgren C, et al.2010]. This hypothesis
is in line with the results presented in our study, showing inhibition of cell
motility ahead of vinculin activation in mAR stimulated Caco2 cells. Notably,
this effect was efficiently reversed by silencing of endogenous vinculin utilizing
short interfering RNAs. Finally, it is worth noting that the effects of TAC on cell
invasion are manifested even after short treatment of cells with this compound
(Fig. 23). Moreover, these effects are still silenced by siRNAs against vinculin
81
(Fig. 23). These results clearly indicate that the inhibitory signals on invasion
are activated early upon mAR stimulation, are dependent on vinculin and are
present in cells well before they commit to the mAR induced apoptotic
program.
82
6. Conclusions
In conclusion, the results presented here add a clear and significant piece of
evidence on the potential anti-tumorigenic role of membrane androgen
receptors.
They indicate that
The functional mAR is expressed not only in hormone-dependent
tumors but also in colon tumors.
mAR conjugates also induced rapid actin and tubulin reorganization.
The activation through steroid albumin conjugates induces potent pro-
apoptotic responses regulated by cytoskeletal rearrangements.
The long term activity of the pro-survival regulators PI-3K, Akt and Bad
is effectively suppressed
mAR Activation Inhibits Cell Motility in Colon Cancer Cells.
The specific molecular target for cell adhesion, vinculin is modulated
upon mAR activation.
Since these molecules may adapt signaling pathways involved in apoptosis,
cell survival and motility [Migliaccio A, et al. 2000, Hanks SK, et al.2003], we
hypothesize that they represent key signaling effectors regulating the mAR
dependent anti-tumorigenic effects reported in our studies. Further
experiments are now needed to address the molecular identity of mAR and to
evaluate the potential role of these signaling targets for the development of
novel anti-tumorigenic strategies based on specific mAR activation. These
receptors may represent specific targets for the development of novel drugs
since their activation drastically regress tumor growth and tumor incidence in
vivo.
83
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Curriculum Vitae
Personal Information:
Family Name: Gu
Given Name: Shuchen
Date of Birth: Aug. 02 1982
Nationality: Chinese
Gender: Female
Marital Status: Single
Education:
2008.2~ Now PhD
Eberhard Karls University Tuebingen
Major: Biology
2005.10 ~ 2008.2 M.S.
Martin-Luther-University Halle-Wittenberg
Hochschule Anhalt University of Applied Science
Major: Biomedical Engineering
2000.9 ~ 2005.7 B.S.
medical college ,Tongji University (Shanghai, China)
Major: clinical medicine
Experience:
2004.1~ 12 Intern
Shanghai East Hospital
Diagnose and treat common illnesses and receive excellent evaluation from doctors and patients.
2002.10~ 12 Volunteer
Tumor Convalescent Center of Shanghai
Help patient with daily care and rehabilitation exercise.
101
Publications obtained during Ph. D Work
Gu S, Papadopoulou N, Nasir O, Föller M, Alevizopoulos K, Lang F,
Stournaras C.Activation of Membrane Androgen Receptors in Colon
Cancer Inhibits the Prosurvival Signals Akt/Bad In Vitro and In Vivo
and Blocks Migration via Vinculin/Actin Signaling.Mol Med. 2011 1-
2;17(1-2):48-58. Epub 2010 Oct 15.
Gu S, Papadopoulou N, Gehring EM, Nasir O, Dimas K, Bhavsar SK,
Föller M, Alevizopoulos K, Lang F, Stournaras C. Functional
membrane androgen receptors in colon tumors trigger pro-apoptotic
responses in vitro and reduce drastically tumor incidence in vivo. Mol
Cancer 2009;8:114.
Wang K*, Gu S*, Nasir O, Föller M, Ackermann TF, Klingel K, Kandolf
R, Kuhl D, Stournaras C, Lang F. SGK1-dependent intestinal tumor
growth in APC-deficient mice. Cell Physiol Biochem. 2010;25(2-
3):271-8 *The authors contributed equally to the manuscript
Shumilina E, Xuan NT, Schmid E, Bhavsar SK, Szteyn K, Gu S, Götz
F, Lang F. Zinc induced apoptotic death of mouse dendritic cells.
Apoptosis. 2010 Jun 22.
Rexhepaj R, Rotte A, Pasham V, Gu S, Kempe DS, Lang F. PI3 kinase
and PDK1 in the regulation of the electrogenic intestinal dipeptide
transport. Cell Physiol Biochem. 2010;25(6):715-22.
Föller M, Mahmud H, Qadri SM, Gu S, Braun M, Bobbala D, Hocher B,
Lang F. Endothelin B receptor stimulation inhibits suicidal erythrocyte
death. FASEB J. 2010 Sep;24(9):3351-9.
Xuan NT, Shumilina E, Gulbins E, Gu S, Götz F, Lang F. Triggering of
dendritic cell apoptosis by xanthohumol. Mol Nutr Food Res. 2010
Jul;54
Bhavsar SK, Föller M, Gu S, Vir S, Shah MB, Bhutani KK, Santani DD,
Lang F. Involvement of the PI3K/AKT pathway in the hypoglycemic
102
effects of saponins from Helicteres isora. J Ethnopharmacol.
2009;126:386-396.
Nasir O, Wang K, Föller M, Gu S, Bhandaru M, Ackermann TF, Boini
KM, Mack A, Klingel K, Amato R, Perrotti N, Kuhl D, Behrens J,
Stournaras C, Lang F. Relative resistance of SGK1 knockout mice
against chemical carcinogenesis. IUBMB Life 2009; 61:768-776.
Föller M, Mahmud H, Gu S, Kucherenko Y, Gehring EM, Shumilina E,
Floride E, Sprengel R, Lang F. Modulation of suicidal erythrocyte
cation channels by an AMPA antagonist. J Cell Mol Med 2009; in
press.
Föller M, Sopjani M, Koka S, Gu S, Mahmud H, Wang K, Floride E,
Schleicher E, Schulz E, Münzel T, Lang F. Regulation of erythrocyte
survival by AMP-activated protein kinase. FASEB J. 2009;23:1072-
1080.
Rexhepaj R *, Rotte A *, Gu S, Pasham V, Wang K, Kempe DS,
Ackermann TF, Föller M, Lang F. Tumor suppressor gene
adenomatous polyposis coli downregulates intestinal transport.
Pflugers Arch. 2011 May;461(5):527-36.
Sopjani M*, Alesutan I*, Dërmaku-Sopjani M, Gu S, Zelenak C, Munoz
C, Velic A, Föller M, Rosenblatt K P, Kuro-o M; Lang F. Regulation of
the Na+/K+ ATPase by Klotho. FEBS Letters. (in publishing)
Bhavsar SK, Gu S, Bobbala D, Lang F. Janus Kinase 3 is Expressed in
Erythrocytes, Phosphorylated Upon Energy Depletion andInvolved in
the Regulation of Suicidal Erythrocyte Death. Cell Physiol Biochem.
(in publishing)
Bhandaru M, Rotte A, Gu S, Föller M, Lang F. Calciuria in mice
carrying a loss of function mutation of the Adenomatous Polyposis
Coli gene. AJP-Regul. (Submitted)
Qadri SM , Mahmud H, Lang E, Gu S, Bobbala D, Zelenak C, Siegfried
A, Föller M, Lang F. Enhanced suicidal erythrocyte death in mice
carrying a loss of function mutation of the Adenomatous Polyposis
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